FcgammaRIIB-Specific Antibodies and Methods of Use Thereof

ABSTRACT

The present invention relates to antibodies or fragments thereof that specifically bind FcγRIIB, particularly human FcγRIIB, with greater affinity than the antibodies or fragments thereof bind FcγRIIA, particularly human FcγRIIA. The present invention also provides the use of an anti-FcγRIIB antibody or an antigen-binding fragment thereof, as a single agent therapy for the treatment, prevention, management, or amelioration of a cancer, preferably a B-cell malignancy, particularly, B-cell chronic lymphocytic leukemia or non-Hodgkin&#39;s lymphoma, an autoimmune disorder, an inflammatory disorder, an IgE-mediated allergic disorder, or one or more symptoms thereof. The invention provides methods of enhancing the therapeutic effect of therapeutic antibodies by administering the antibodies of the invention to enhance the effector function of the therapeutic antibodies. The invention also provides methods of enhancing efficacy of a vaccine composition by administering the antibodies of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/123,816, filed Sep. 6, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/331,360, filed Jul. 15, 2014, which is adivisional of U.S. patent application Ser. No. 11/768,852, filed Jun.26, 2007, which application claims the benefit of U.S. ProvisionalPatent Application Ser. No. 60/816,688, filed Jun. 26, 2006, thedisclosures of which are incorporated herein by reference in theirentireties and to which priority is claimed.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(filed name: 13010015C_ST25, created Jul. 13, 2021, and having a size of7118 bytes), which file is herein incorporated by reference in itsentirety.

1. FIELD OF THE INVENTION

The present invention relates to antibodies or fragments thereof thatspecifically bind FcγRIIB, particularly human FcγRIIB, with greateraffinity than said antibodies or fragments thereof bind FcγRIIA,particularly human FcγRIIA. The present invention also encompasses theuse of an anti-FcγRIIB antibody or an antigen-binding fragment thereof,as a single agent therapy for the treatment, prevention, management, oramelioration of a cancer, preferably a B-cell malignancy, particularly,B-cell chronic lymphocytic leukemia or non-Hodgkin's lymphoma, anautoimmune disorder, an inflammatory disorder, an IgE-mediated allergicdisorder, or one or more symptoms thereof. The present invention alsoencompasses the use of an anti-FcγRIIB antibody or an antigen-bindingfragment thereof, in combination with other cancer therapies. Thepresent invention provides pharmaceutical compositions comprising ananti-FcγRIIB antibody or an antigen-binding fragment thereof, in amountseffective to prevent, treat, manage, or ameliorate a cancer, such as aB-cell malignancy, an autoimmune disorder, an inflammatory disorder, anIgE-mediated allergic disorder, or one or more symptoms thereof. Theinvention further provides methods of enhancing the therapeutic effectof therapeutic antibodies by administering the antibodies of theinvention to enhance the effector function of the therapeuticantibodies. The invention also provides methods of enhancing efficacy ofa vaccine composition by administering the antibodies of the inventionwith a vaccine composition.

2. BACKGROUND OF THE INVENTION 2.1 Fc Receptors and their Roles in theImmune System

The interaction of antibody-antigen complexes with cells of the immunesystem results in a wide array of responses, ranging from effectorfunctions such as antibody-dependent cell-mediated cytotoxicity, mastcell degranulation, and phagocytosis to immunomodulatory signals such asregulating lymphocyte proliferation and antibody secretion. All theseinteractions are initiated through the binding of the Fc domain ofantibodies or immune complexes to specialized cell surface receptors onhematopoietic cells. The diversity of cellular responses triggered byantibodies and immune complexes results from the structuralheterogeneity of Fc receptors. Fc receptors share structurally relatedligand binding domains which presumably mediate intracellular signaling.

The Fc receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fc portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe a chain of the Fc receptor. Fc receptors are defined by theirspecificity for immunoglobulin subtypes. Fc receptors for IgG arereferred to as FcγR, for IgE as FcεR, and for IgA as FcαR. Differentaccessory cells bear Fc receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (reviewed by Ravetch J. V. et al. 1991,Annu. Rev. Immunol. 9: 457-92; Gerber J. S. et al. 2001 Microbes andInfection, 3: 131-139; Billadeau D. D. et al. 2002, The Journal ofClinical Investigation, 2(109): 161-1681; Ravetch J. V. et al. 2000,Science, 290: 84-89; Ravetch J. V. et al., 2001 Annu. Rev. Immunol.19:275-90; Ravetch J. V. 1994, Cell, 78(4): 553-60). The different Fcreceptors, the cells that express them, and their isotype specificity issummarized in Table 1 (adapted from Immunobiology: The Immune System inHealth and Disease, 4^(th) ed. 1999, Elsevier Science Ltd/GarlandPublishing, New York).

Fcγ Receptors

Each member of this family is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are three known FcγRs,designated FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). The threereceptors are encoded by distinct genes; however, the extensive homologybetween the three family members suggest they arose from a commonprogenitor perhaps by gene duplication. This invention specificallyfocuses on FcγRII(CD32).

FcγRII(CD32)

FcγRII proteins are 40 KDa integral membrane glycoproteins which bindonly the complexed IgG due to a low affinity for monomeric Ig (10⁶ M¹).This receptor is the most widely expressed FcγR, present on allhematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRT. There are three humanFcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG inaggregates or immune complexes.

Distinct differences within the cytoplasmic domains of FcγRII-A (CD32A)and FcγRII-B (CD32B) create two functionally heterogeneous responses toreceptor ligation. The fundamental difference is that the A isoforminitiates intracellular signaling leading to cell activation such asphagocytosis and respiratory burst, whereas the B isoform initiatesinhibitory signals, e.g., inhibiting B-cell activation.

Signaling Through FcγRs

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called immunoreceptor tyrosine based activation motifs (ITAMs)or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRT,FcγRIIA, FcγRIIIA, whereas ITIM-containing complexes only includeFcγRIIB

Human neutrophils express the FcγRIIA gene. FcγRIIA clustering viaimmune complexes or specific antibody cross-linking serves to aggregateITAMs along with receptor-associated kinases which facilitate ITAMphosphorylation. ITAM phosphorylation serves as a docking site for Sykkinase, activation of which results in activation of downstreamsubstrates (e.g., PI₃K). Cellular activation leads to release ofproinflammatory mediators.

The FcγRIIB gene is expressed on B lymphocytes; its extracellular domainis 96% identical to FcγRIIA and binds IgG complexes in anindistinguishable manner. The presence of an ITIM in the cytoplasmicdomain of FcγRIIB defines this inhibitory subclass of FcγR. Recently themolecular basis of this inhibition was established. When colligatedalong with an activating FcγR, the ITIM in FcγRIIB becomesphosphorylated and attracts the SH2 domain of the inositol polyphosphate5′-phosphatase (SHIP), which hydrolyzes phosphoinositol messengersreleased as a consequence of ITAM-containing FcγR-mediated tyrosinekinase activation, consequently preventing the influx of intracellularCa⁺⁺. Thus, crosslinking of FcγRIIB dampens the activating response toFcγR ligation and inhibits cellular responsiveness. B cell activation, Bcell proliferation and antibody secretion is thus aborted.

TABLE 1 Receptors for the Fc Regions of Immunoglobulin Isotypes ReceptorFcγRI FcγRII-A FcγRII-B2 FcγRII-BI FcγRIII FcαRI (CD64) (CD32) (CD32)(CD32) (CD16) FcεRI (CD89) Binding IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgGl,IgA2 10⁸ M⁻¹ 2 × 10⁶ M⁻¹ 2 × 10⁶ M⁻¹ 2 × 10⁶ M⁻¹ 5 × 10⁵ M⁻¹ 10¹⁰ M⁻¹10⁷ M⁻¹ Cell Type Macrophages Macrophages Macrophages B cells NK cellsMast cells Macrophages Neutrophils Neutrophils Neutrophils Mast cellsEosinophil Eosinophil Neutropils Eosinophils Eosinophils Eosinophilsmacrophages Basophils Eosinophils Dendritic cells Dendritic cellsNeutrophils Platelets Mast Cells Langerhan cells Effect of Uptake UptakeUptake No uptake Induction of Secretion of Uptake Ligation StimulationGranule Inhibition of Inhibition of Killing granules Induction ofActivation of release Stimulation Stimulation killing respiratory burstInduction of killing

2.2 Diseases of Relevance

2.2.1 Cancer

A neoplasm, or tumor, is a neoplastic mass resulting from abnormaluncontrolled cell growth which can be benign or malignant. Benign tumorsgenerally remain localized. Malignant tumors are collectively termedcancers. The term “malignant” generally means that the tumor can invadeand destroy neighboring body structures and spread to distant sites tocause death (for review, see Robbins and Angell, 1976, Basic Pathology,2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-122). Cancer can arisein many sites of the body and behave differently depending upon itsorigin. Cancerous cells destroy the part of the body in which theyoriginate and then spread to other part(s) of the body where they startnew growth and cause more destruction.

More than 1.2 million Americans develop cancer each year. Cancer is thesecond leading case of death in the United States and if current trendscontinue, cancer is expected to be the leading cause of the death by theyear 2010. Lung and prostate cancer are the top cancer killers for menin the United States. Lung and breast cancer are the top cancer killersfor women in the United States. One in two men in the United States willbe diagnosed with cancer at some time during his lifetime. One in threewomen in the United States will be diagnosed with cancer at some timeduring her lifetime. A cure for cancer has yet to be found. Currenttreatment options, such as surgery, chemotherapy and radiationtreatment, are often times either ineffective or present serious sideeffects.

2.2.1.1 B-Cell Malignancies

B cell malignancies, including, but not limited to, B-cell lymphomas andleukemias, are neoplastic diseases with significant incidence in theUnited States. There are approximately 55,000 new lymphoma cases of peryear in the U.S. (1998 data), with an estimated 25,000 deaths per year.This represents 4% of cancer incidence and 4% of all cancer-relateddeaths in the U.S. population. The revised European-Americanclassification of lymphoid neoplasms (1994 REAL classification, modified1999) grouped lymphomas based on their origin as either B cell lineagelymphoma, T cell lineage lymphoma, or Hodgkin's lymphoma. Lymphoma ofthe B cell lineage is the most common type of non-Hodgkin's lymphoma(NHL) diagnosed in the U.S. (Williams, Hematology 6^(th) ed. (Beutler etal. Ed.), McGraw Hill 2001).

Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterizedby the accumulation of small, mature-appearing lymphocytes in the blood,marrow, and lymphoid tissues. CLL has an incidence of 2.7 cases per100,000 in the U.S. The risk increases progressively with age,particularly in men. It accounts for 0.8% of all cancers and is the mostcommon adult leukemia, responsible for 30% of all leukemias. In nearlyall cases (>98%) the diseased cells belong to the B lymphocyte lineage.A non-leukemic variant, small lymphocytic lymphoma, constitutes 5-10% ofall lymphomas, has histological, morphological and immunologicalfeatures indistinguishable from that of involved lymph nodes in patientswith B-CLL (Williams, 2001).

The natural history of chronic lymphocytic leukemia falls into severalphases. In the early phase, chronic lymphocytic leukemia is an indolentdisease, characterized by the accumulation of small, mature,functionally-incompetent malignant B-cells having a lengthened lifespan. Eventually, the doubling time of the malignant B-cells decreasesand patients become increasingly symptomatic. While treatment withchemotherapeutic agents can provide symptomatic relief, the overallsurvival of the patients is only minimally extended. The late stages ofchronic lymphocytic leukemia are characterized by significant anemiaand/or thrombocytopenia. At this point, the median survival is less thantwo years (Foon et al., 1990, Annals Int. Medicine 113:525). Due to thevery low rate of cellular proliferation, chronic lymphocytic leukemia isresistant to treatment with chemotherapeutic agents.

Recently, gene expression studies have identified several genes that maybe up regulated in lymphoproliferative disorders. One molecule thoughtto be over-expressed in patients with B-cell chronic lymphocyticleukemia (B-CLL) and in a large fraction of non-Hodgkin lymphomapatients is CD32B (Alizadeh et al., 2000, Nature 403:503-511; Rosenwaldet al., 2001, J. Exp. Med. 184:1639-1647). However, the role of CD32B isB-CLL is unclear since one report demonstrates that CD32B was expressedon a low percentage of B-CLL cells and at a low density (Damle et al.,2002, Blood 99:4087-4093). CD32B is a B cell lineage surface antigen,whose over-expression in B cell neoplasia makes it a suitable target fortherapeutic antibodies. In addition, CD32B belongs to the category ofinhibitory receptors, whose ligation delivers a negative signal.Therefore, antibodies directed against CD32B could function to eliminatetumor cells by mechanisms that include complement dependent cytotoxicity(CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), but alsotriggering an apoptotic signal. The high homology of CD32B with itscounterpart, CD32A, an activating Fcγ receptor, has thus far hamperedthe generation of antibodies that selectively recognize one but not theother form of the molecule.

2.2.1.2 Cancer Therapy

Currently, cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (See, for example, Stockdale, 1998, “Principles of CancerPatient Management”, in Scientific American: Medicine, vol. 3,Rubenstein and Federman, eds., Chapter 12, Section IV). Recently, cancertherapy could also involve biological therapy or immunotherapy. All ofthese approaches pose significant drawbacks for the patient. Surgery,for example, may be contraindicated due to the health of the patient ormay be unacceptable to the patient. Additionally, surgery may notcompletely remove the neoplastic tissue. Radiation therapy is onlyeffective when the neoplastic tissue exhibits a higher sensitivity toradiation than normal tissue, and radiation therapy can also oftenelicit serious side effects. Hormonal therapy is rarely given as asingle agent and although can be effective, is often used to prevent ordelay recurrence of cancer after other treatments have removed themajority of the cancer cells. Biological therapies/immunotherapies arelimited in number and may produce side effects such as rashes orswellings, flu-like symptoms, including fever, chills and fatigue,digestive tract problems or allergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A significant majority ofcancer chemotherapeutics act by inhibiting DNA synthesis, eitherdirectly, or indirectly by inhibiting the biosynthesis of thedeoxyribonucleotide triphosphate precursors, to prevent DNA replicationand concomitant cell division (See, for example, Gilman et al., Goodmanand Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed.(Pergamom Press, New York, 1990)). These agents, which includealkylating agents, such as nitrosourea, anti-metabolites, such asmethotrexate and hydroxyurea, and other agents, such as etoposides,camptothecins, bleomycin, doxorubicin, daunorubicin, etc., although notnecessarily cell cycle specific, kill cells during S phase because oftheir effect on DNA replication. Other agents, specifically colchicineand the vinca alkaloids, such as vinblastine and vincristine, interferewith microtubule assembly resulting in mitotic arrest. Chemotherapyprotocols generally involve administration of a combination ofchemotherapeutic agents to increase the efficacy of treatment.

Despite the availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks (See, for example, Stockdale, 1998,“Principles Of Cancer Patient Management” in Scientific AmericanMedicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10).Almost all chemotherapeutic agents are toxic, and chemotherapy causessignificant, and often dangerous, side effects, including severe nausea,bone marrow depression, immunosuppression, etc. Additionally, even withadministration of combinations of chemotherapeutic agents, many tumorcells are resistant or develop resistance to the chemotherapeuticagents. In fact, those cells resistant to the particularchemotherapeutic agents used in the treatment protocol often prove to beresistant to other drugs, even those agents that act by mechanismsdifferent from the mechanisms of action of the drugs used in thespecific treatment; this phenomenon is termed pleiotropic drug ormultidrug resistance. Thus, because of drug resistance, many cancersprove refractory to standard chemotherapeutic treatment protocols.

B cell malignancy is generally treated with single agent chemotherapy,combination chemotherapy and/or radiation therapy. These treatments canreduce morbidity and/or improve survival, albeit they carry significantside effects. The response of B-cell malignancies to various forms oftreatment is mixed. For example, in cases in which adequate clinicalstaging of non-Hodgkin's lymphoma is possible, field radiation therapycan provide satisfactory treatment. Certain patients, however, fail torespond and disease recurrence with resistance to treatment ensues withtime, particularly with the most aggressive variants of the disease.About one-half of the patients die from the disease (Devesa et al.,1987, J. Nat'l Cancer Inst. 79:701).

Investigational therapies for the treatment of refractory B cellneoplasia include autologous and allogeneic bone marrow or stem celltransplantation and gene therapies. Recently, immunotherapy usingmonoclonal antibodies to target B-cell specific antigens has beenintroduced in the treatment of B cell neoplasia. The use of monoclonalantibodies to direct radionuclides, toxins, or other therapeutic agentsoffers the possibility that such agents can be delivered selectively totumor sites, thus limiting toxicity to normal tissues.

There is a significant need for alternative cancer treatments,particularly for treatment of cancer that has proved refractory tostandard cancer treatments, such as surgery, radiation therapy,chemotherapy, and hormonal therapy. A promising alternative isimmunotherapy, in which cancer cells are specifically targeted by cancerantigen-specific antibodies. Major efforts have been directed atharnessing the specificity of the immune response, for example,hybridoma technology has enabled the development of tumor selectivemonoclonal antibodies (See Green M. C. et al., 2000 Cancer Treat Rev.,26: 269-286; Weiner L M, 1999 Semin Oncol. 26(suppl. 14):43-51), and inthe past few years, the Food and Drug Administration has approved thefirst MAbs for cancer therapy: Rituxin (anti-CD20) for non-Hodgkin'sLymphoma, Campath (anti-CD52) for B-cell chronic lymphocytic leukemia(B-CLL) and Herceptin [anti-(c-erb-2/HER-2)] for metastatic breastcancer (Suzanne A. Eccles, 2001, Breast Cancer Res., 3: 86-90). NHL andB-CLL are two of the most common forms of B cell neoplasia. Theseantibodies have demonstrated clinical efficacy, but their use is notwithout side effects. The potency of antibody effector function, e.g.,to mediate antibody-dependent cell-mediated cytotoxicity (“ADCC”) is anobstacle to such treatment. Furthermore, with Rituxan and Campath, atleast half the patients fail to respond and a fraction of responders maybe refractory to subsequent treatments.

There is a need for alternative therapies for cancer, particularly,B-cell malignancies, especially for patients that are refractory forstandard cancer treatments and new immunotherapies such as Rituxan.

2.2.2 Inflammatory Diseases and Autoimmune Diseases

Inflammation is a process by which the body's white blood cells andchemicals protect our bodies from infection by foreign substances, suchas bacteria and viruses. It is usually characterized by pain, swelling,warmth and redness of the affected area. Chemicals known as cytokinesand prostaglandins control this process, and are released in an orderedand self-limiting cascade into the blood or affected tissues. Thisrelease of chemicals increases the blood flow to the area of injury orinfection, and may result in the redness and warmth. Some of thechemicals cause a leak of fluid into the tissues, resulting in swelling.This protective process may stimulate nerves and cause pain. Thesechanges, when occurring for a limited period in the relevant area, workto the benefit of the body.

In autoimmune and/or inflammatory disorders, the immune system triggersan inflammatory response when there are no foreign substances to fightand the body's normally protective immune system causes damage to itsown tissues by mistakenly attacking self. There are many differentautoimmune disorders which affect the body in different ways. Forexample, the brain is affected in individuals with multiple sclerosis,the gut is affected in individuals with Crohn's disease, and thesynovium, bone and cartilage of various joints are affected inindividuals with rheumatoid arthritis. As autoimmune disorders progressdestruction of one or more types of body tissues, abnormal growth of anorgan, or changes in organ function may result. The autoimmune disordermay affect only one organ or tissue type or may affect multiple organsand tissues. Organs and tissues commonly affected by autoimmunedisorders include red blood cells, blood vessels, connective tissues,endocrine glands (e.g., the thyroid or pancreas), muscles, joints, andskin. Examples of autoimmune disorders include, but are not limited to,Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1diabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, dermatomyositis, lupuserythematosus, multiple sclerosis, autoimmune inner ear diseasemyasthenia gravis, Reiter's syndrome, Graves disease, autoimmunehepatitis, familial adenomatous polyposis and ulcerative colitis.

Rheumatoid arthritis (RA) and juvenile rheumatoid arthritis are types ofinflammatory arthritis. Arthritis is a general term that describesinflammation in joints. Some, but not all, types of arthritis are theresult of misdirected inflammation. Besides rheumatoid arthritis, othertypes of arthritis associated with inflammation include the following:psoriatic arthritis, Reiter's syndrome, ankylosing spondylitisarthritis, and gouty arthritis. Rheumatoid arthritis is a type ofchronic arthritis that occurs in joints on both sides of the body (suchas both hands, wrists or knees). This symmetry helps distinguishrheumatoid arthritis from other types of arthritis. In addition toaffecting the joints, rheumatoid arthritis may occasionally affect theskin, eyes, lungs, heart, blood or nerves.

Rheumatoid arthritis affects about 1% of the world's population and ispotentially disabling. There are approximately 2.9 million incidences ofrheumatoid arthritis in the United States. Two to three times more womenare affected than men. The typical age that rheumatoid arthritis occursis between 25 and 50. Juvenile rheumatoid arthritis affects 71,000 youngAmericans (aged eighteen and under), affecting six times as many girlsas boys.

Rheumatoid arthritis is an autoimmune disorder where the body's immunesystem improperly identifies the synovial membranes that secrete thelubricating fluid in the joints as foreign. Inflammation results, andthe cartilage and tissues in and around the joints are damaged ordestroyed. In severe cases, this inflammation extends to other jointtissues and surrounding cartilage, where it may erode or destroy boneand cartilage and lead to joint deformities. The body replaces damagedtissue with scar tissue, causing the normal spaces within the joints tobecome narrow and the bones to fuse together. Rheumatoid arthritiscreates stiffness, swelling, fatigue, anemia, weight loss, fever, andoften, crippling pain. Some common symptoms of rheumatoid arthritisinclude joint stiffness upon awakening that lasts an hour or longer;swelling in a specific finger or wrist joints; swelling in the softtissue around the joints; and swelling on both sides of the joint.Swelling can occur with or without pain, and can worsen progressively orremain the same for years before progressing.

The diagnosis of rheumatoid arthritis is based on a combination offactors, including: the specific location and symmetry of painfuljoints, the presence of joint stiffness in the morning, the presence ofbumps and nodules under the skin (rheumatoid nodules), results of X-raytests that suggest rheumatoid arthritis, and/or positive results of ablood test called the rheumatoid factor. Many, but not all, people withrheumatoid arthritis have the rheumatoid-factor antibody in their blood.The rheumatoid factor may be present in people who do not haverheumatoid arthritis. Other diseases can also cause the rheumatoidfactor to be produced in the blood. That is why the diagnosis ofrheumatoid arthritis is based on a combination of several factors andnot just the presence of the rheumatoid factor in the blood.

The typical course of the disease is one of persistent but fluctuatingjoint symptoms, and after about 10 years, 90% of sufferers will showstructural damage to bone and cartilage. A small percentage will have ashort illness that clears up completely, and another small percentagewill have very severe disease with many joint deformities, andoccasionally other manifestations of the disease. The inflammatoryprocess causes erosion or destruction of bone and cartilage in thejoints. In rheumatoid arthritis, there is an autoimmune cycle ofpersistent antigen presentation, T-cell stimulation, cytokine secretion,synovial cell activation, and joint destruction. The disease has a majorimpact on both the individual and society, causing significant pain,impaired function and disability, as well as costing millions of dollarsin healthcare expenses and lost wages. (See, for example, the NIHwebsite and the NIAID website).

Currently available therapy for arthritis focuses on reducinginflammation of the joints with anti-inflammatory or immunosuppressivemedications. The first line of treatment of any arthritis is usuallyanti-inflammatories, such as aspirin, ibuprofen and Cox-2 inhibitorssuch as celecoxib and rofecoxib. “Second line drugs” include gold,methotrexate and steroids. Although these are well-establishedtreatments for arthritis, very few patients remit on these lines oftreatment alone. Recent advances in the understanding of thepathogenesis of rheumatoid arthritis have led to the use of methotrexatein combination with antibodies to cytokines or recombinant solublereceptors. For example, recombinant soluble receptors for tumor necrosisfactor (TNF)-α have been used in combination with methotrexate in thetreatment of arthritis. However, only about 50% of the patients treatedwith a combination of methotrexate and anti-TNF-α agents such asrecombinant soluble receptors for TNF-α show clinically significantimprovement. Many patients remain refractory despite treatment.Difficult treatment issues still remain for patients with rheumatoidarthritis. Many current treatments have a high incidence of side effectsor cannot completely prevent disease progression. So far, no treatmentis ideal, and there is no cure. Novel therapeutics are needed that moreeffectively treat rheumatoid arthritis and other autoimmune disorders.

2.2.3 Allergy

Immune-mediated allergic (hypersensitivity) reactions are classifiedinto four types (I-IV) according to the underlying mechanisms leading tothe expression of the allergic symptoms. Type I allergic reactions arecharacterized by IgE-mediated release of vasoactive substances such ashistamine from mast cells and basophils. The release of these substancesand the subsequent manifestation of allergic symptoms are initiated bythe cross-linking of allergen-bound IgE to its receptor on the surfaceof mast cells and basophils. In individuals suffering from type Iallergic reactions, exposure to an allergen for a second time leads tothe production of high levels of IgE antibodies specific for theallergen as a result of the involvement of memory B and T cells in the3-cell interaction required for IgE production. The high levels of IgEantibodies produced cause an increase in the cross-linking of IgEreceptors on mast cells and basophils by allergen-bound IgE, which inturn leads to the activation of these cells and the release of thepharmacological mediators that are responsible for the clinicalmanifestations of type I allergic diseases.

Two receptors with differing affinities for IgE have been identified andcharacterized. The high affinity receptor (FcεRI) is expressed on thesurface of mast cells and basophils. The low affinity receptor(FcεRII/CD23) is expressed on many cell types including B cells, Tcells, macrophages, eosinophils and Langerhan cells. The high affinityIgE receptor consists of three subunits (alpha, beta and gamma chains).Several studies demonstrate that only the alpha chain is involved in thebinding of IgE, whereas the beta and gamma chains (which are eithertransmembrane or cytoplasmic proteins) are required for signaltransduction events. The identification of IgE structures required forIgE to bind to the FcεRI on mast cells and basophils is of utmostimportance in devising strategies for treatment or prevention ofIgE-mediated allergies. For example, the elucidation of the IgEreceptor-binding site could lead to the identification of peptides orsmall molecules that block the binding of IgE to receptor-bearing cellsin vivo.

Currently, IgE-mediated allergic reactions are treated with drugs suchas antihistamines and corticosteroids which attempt to alleviate thesymptoms associated with allergic reactions by counteracting the effectsof the vasoactive substances released from mast cells and basophils.High doses of antihistamines and corticosteroids have deleterious sideeffects (e.g., central nervous system disturbance, constipation, etc).Thus, other methods for treating type I allergic reactions are needed.

One approach to the treatment of type I allergic disorders has been theproduction of monoclonal antibodies which react with soluble (free) IgEin serum, block IgE from binding to its receptor on mast cells andbasophils, and do not bind to receptor-bound IgE (i.e., they arenon-anaphylactogenic). Two such monoclonal antibodies are in advancedstages of clinical development for treatment of IgE-mediated allergicreactions (see, e.g., Chang, T. W., 2000, Nature Biotechnology18:157-62).

One of the most promising treatments for IgE-mediated allergic reactionsis the active immunization against appropriate non-anaphylactogenicepitopes on endogenous IgE. Stanworth et al. (U.S. Pat. No. 5,601,821)described a strategy involving the use of a peptide derived from theCεH4 domain of the human IgE coupled to a heterologous carrier proteinas an allergy vaccine. However, this peptide has been shown not toinduce the production of antibodies that react with native soluble IgE.Further, Hellman (U.S. Pat. No. 5,653,980) proposed anti-IgE vaccinecompositions based on fusion of full length CεH2-CεH3 domains(approximately 220 amino acid long) to a foreign carrier protein.However, the antibodies induced by the anti-IgE vaccine compositionsproposed in Hellman will most likely it result in anaphylaxis sinceantibodies against some portions of the CεH2 and CεH3 domains of the IgEmolecule have been shown to cross-link the IgE receptor on the surfaceof mast cell and basophils and lead to production of mediators ofanaphylaxis (See, e.g., Stadler et al., 1993, Int. Arch. Allergy andImmunology 102:121-126). Therefore, a need remains for treatment ofIgE-mediated allergic reactions which do not induce anaphylacticantibodies.

The significant concern over induction of anaphylaxis has resulted inthe development of another approach to the treatment of type I allergicdisorders consisting of mimotopes that could induce the production ofanti-IgE polyclonal antibodies when administered to animals (See, e.g.,Rudolf, et al., 1998, Journal of Immunology 160:3315-3321). Kricek etal. (International Publication No. WO 97/31948) screened phage-displayedpeptide libraries with the monoclonal antibody BSWI7 to identify peptidemimotopes that could mimic the conformation of the IgE receptor binding.These mimotopes could presumably be used to induce polyclonal antibodiesthat react with free native IgE, but not with receptor-bound IgE as wellas block IgE from binding to its receptor. Kriek et al. disclosedpeptide mimotopes that are not homologous to any part of the IgEmolecule and are thus different from peptides disclosed in the presentinvention.

As evidenced by a survey of the art, there remains a need for enhancingthe therapeutic efficacy of current methods of treating or preventingdisorders such as cancer, autoimmune disease, inflammatory disorder, orallergy. In particular, there is a need for enhancing the effectorfunction, particularly, the cytotoxic effect of therapeutic antibodiesused in treatment of cancer. The current state of the art is alsolacking in treating or preventing allergy disorders (e.g., either byantibody therapy or vaccine therapy).

3. SUMMARY OF THE INVENTION

The extracellular domains of FcγRIIA and FcγRIIB are 95% identical andthus they share numerous epitopes. However, FcγRIIA and FcγRIIB exhibitvery different activities. The fundamental difference is that theFcγRIIA initiates intracellular signaling leading to cell activationsuch as phagocytosis and respiratory burst, whereas the FcγRIIBinitiates inhibitory signaling. In view of their distinctive activitiesand role in modulating immune responses, such antibodies that recognizenative FcγRIIB, and not native FcγRIIA, are needed. The presentinvention is based, in part, on the discovery of such FcγRIIB-specificantibodies.

The invention relates to an isolated antibody or a fragment thereof thatspecifically binds FcγRIIB, particularly human FcγRIIB, moreparticularly native human FcγRIIB, with a greater affinity than saidantibody or a fragment thereof binds FcγRIIA, particularly humanFcγRIIA, more particularly native human FcγRIIA. Preferably theantibodies of the invention bind the extracellular domain of nativehuman FcγRIIB In certain embodiments of the invention, the antibody or afragment thereof binds FcγRIIB with at least 2 times greater affinitythan said antibody or a fragment thereof binds FcγRIIA. In otherembodiments of the invention, the antibody or a fragment thereof bindsFcγRIIB with at least 4 times, at least 6 times, at least 8 times, atleast 10 times, at least 100 times, at least 1000 times, at least 10⁴,at least 10⁵, at least 10⁶, at least 10⁷, or at least 10⁸ times greateraffinity than said antibody or a fragment thereof binds FcγRIIA. In apreferred embodiment, said antibody or a fragment thereof binds FcγRIIBwith 100 times, 1000 times, 10⁴ times, 10⁵ times, 10⁶ times, 10⁷ times,or 10⁸ times greater affinity than said antibody or a fragment thereofbinds FcγRIIA. Preferably, these binding affinities are determined withthe monomeric IgG, and not the aggregated IgG, and binding is via thevariable domain (e.g., Fab fragments of the antibodies have bindingcharacteristic similar to the full immunoglobulin molecule).

In one embodiment, the FcγRIIB-specific antibody in accordance with theinvention is not the monoclonal antibody designated KB61, as disclosedin Pulford et al., 1986 (Immunology, 57: 71-76) or the monoclonalantibody designated MAbII8D2 as disclosed in Weinrich et al., 1996,(Hybridoma, 15(2):109-6). In a specific embodiment, the FcγRIIB-specificantibody of the invention does not bind to the same epitope and/or doesnot compete for binding with the monoclonal antibody KB61 or themonoclonal antibody MAbII8D2. Preferably, the FcγRIIB-specific antibodyof the invention does not bind the amino acid sequenceSer-Asp-Pro-Asn-Phe-Ser-Ile (SEQ ID NO: 20) corresponding to amino acidpositions 135-141 of FcγRIIb2 isoform.

The invention relates to an isolated antibody or a fragment thereof thatspecifically binds FcγRIIB with a greater affinity than said antibody ora fragment thereof binds FcγRIIA, as determined by any standard methodknown in the art for assessing specificities. The invention relates toan isolated antibody or a fragment thereof that specifically bindsFcγRIIB with a greater affinity than said antibody or a fragment thereofbinds FcγRIIA, as determined, for example, by western blot, BIAcore orradioimmunoassay. The invention relates to an isolated antibody or afragment thereof that specifically binds FcγRIIB with a greater affinitythan said antibody or a fragment thereof binds FcγRIIA, as determined inan ELISA assay, in the linear range for FcγRIIB binding. In oneembodiment of the invention, the invention relates to an isolatedantibody, or a fragment thereof that specifically binds FcγRIIB,produced in mammalian system, with a greater affinity than said antibodyor a fragment thereof binds FcγRIIA, as determined in an ELISA assay.

In a particular embodiment, the invention relates to an isolatedantibody or a fragment thereof that specifically binds FcγRIIB with agreater affinity than said antibody or a fragment thereof binds FcγRIIA,and the constant domain of said antibody further has an enhancedaffinity for at least one or more Fc activation receptors. In yetanother specific embodiment, said Fc activation receptor is FcγRIII.

In one embodiment of the invention said antibody or a fragment thereofblocks the IgG binding site of FcγRIIB and blocks the binding ofaggregated labeled IgGs to FcγRIIB in, for example, a blocking ELISAassay. In one particular embodiment, said antibody or a fragment thereofblocks the binding of aggregated labeled IgGs in an ELISA blocking assayby at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9%. In yet anotherparticular embodiment, the antibody or a fragment thereof completelyblocks the binding of said aggregated labeled IgG in said ELISA assay.

In another embodiment of the invention, said antibody or a fragmentthereof blocks the IgG binding site of FcγRIIB and blocks the binding ofaggregated labeled IgG to FcγRIIB, as determined by a double-stainingFACS assay.

The invention encompasses the use of antibodies that modulate (i.e.,agonize or antagonize) the activity of FcγRIIB In one embodiment of theinvention, the antibodies of the invention agonize at least one activityof FcγRIIB, i.e., elicit signaling. Although not intending to be boundby any mechanism of action, agonistic antibodies of the invention maymimic clustering of FcγRIIB leading to dampening of the activatingresponse to FcγR ligation and inhibition of cellular responsiveness.

In another embodiment of the invention, the antibodies of the inventionantagonize at least one activity of FcγRIIB, i.e., block signaling. Forexample, the antibodies of the invention block the binding of aggregatedIgGs to FcγRIIB

The invention provides antibodies that inhibit FcεRI-induced mast cellactivation. The invention further provides anti-FcγRIIB antibodies thatinhibit FcγRIIA-mediated macrophage activation in monocytic cells. Theinvention also provides anti-FcγRIIB antibodies that inhibit B-cellreceptor mediated signaling.

In one particular embodiment, the anti-FcγRIIB antibodies block theligand binding site of FcγRIIB In a further specific embodiment, theblocking activity can block the negative regulation ofimmune-complex-triggered activation and consequently enhance the immuneresponse. In a further specific embodiment, the enhanced immune responseis an increase in antibody-dependent cellular response. In anotherspecific embodiment, the anti-FcγRIIB antibodies of the invention blockcrosslinking of FcγRIIB receptors to B cell and/or Fc receptors, leadingto B cell, mast cell, dendritic cell, or macrophage activation.

The present invention encompasses methods for the production ofantibodies of the invention or fragments thereof, particularly for theproduction of novel monoclonal antibodies (“MAb”) with higherspecificities for FcγRIIB relative to FcγRIIA. The antibodies of theinvention or fragments thereof can be produced by any method known inthe art for the production of antibodies, in particular, by secretionfrom cultured hybridoma cells, chemical synthesis or by recombinantexpression techniques known in the art. In one specific embodiment, theinvention relates to a method for recombinantly producing aFcγRIIB-specific antibody, said method comprising: (i) culturing underconditions suitable for the expression of said antibody in a medium, ahost cell containing a first nucleic acid molecule, operably linked to aheterologous promoter and a second nucleic acid operably linked to thesame or a different heterologous promoter, said first nucleic acid andsecond nucleic acid encoding a heavy chain and a light chain,respectively, of an antibody or a fragment thereof that specificallybinds FcγRIIB with greater affinity than said antibody or a fragmentthereof binds FcγRIIA; and (ii) recovery of said antibody from saidmedium. In another embodiment, the invention provides a method forproducing FcγRIIB monoclonal antibodies that specifically bind FcγRIIB,particularly human FcγRIIB, with a greater affinity than said monoclonalantibodies bind FcγRIIA, particularly human FcγRIIA, said methodcomprising:

(a) immunizing one or more FcγRIIA transgenic mice with purified FcγRIIBor an immunogenic fragment thereof; (b) producing hybridoma cells linesfrom spleen cells of said one or more mice; (c) screening said hybridomacell lines for one or more hybridoma cell lines that produce antibodiesthat specifically bind FcγRIIB with a greater affinity than theantibodies bind FcγRIIA. The invention encompasses any antibody producedby said method. In one specific embodiment, the invention provides amethod for producing FcγRIIB monoclonal antibodies that specificallybind FcγRIIB, particularly human FcγRIIB, with a greater affinity thansaid monoclonal antibodies bind FcγRIIA, particularly human FcγRIIA,said method comprising: (a) immunizing one or more FcγRIIA transgenicmice with purified FcγRIIB or an immunogenic fragment thereof; (b)booster immunizing said mice for a time sufficient to elicit an immuneresponse; (c) producing hybridoma cells lines from spleen cells of saidone or more mice; (d) screening said hybridoma cell lines for one ormore hybridoma cell lines that produce antibodies that specifically bindFcγRIIB with a greater affinity than the antibodies bind FcγRIIA. In apreferred embodiment, said mice are booster immunized at least fourtimes over a period of four months. In one embodiment of the invention,said mice are immunized with purified FcγRIIB, which has been mixed withadjuvants known in the art to enhance immune response in said mice. Inone particular embodiment of the invention, said immunogenic fragment isthe soluble extracellular domain of FcγRIIB The hybridoma cell lines canbe screened using standard techniques known in the art (e.g., ELISA).

In certain embodiments of the invention, the anti-FcγRIIB antibodies aremonoclonal antibodies, synthetic antibodies, recombinantly producedantibodies, multispecific antibodies, human antibodies, chimericantibodies, camelized antibodies, single-chain Fvs (scFv), single chainantibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs(sdFv), intrabodies, or epitope-binding fragments of any of the above.

Preferably, the antibodies of the invention are monoclonal antibodies,and more preferably, humanized or human antibodies. In one specificpreferred embodiment, the antibodies of the invention bind to theextracellular domain of human FcγRIIB, particularly native human FcγRIIBIn another specific embodiment, the antibodies of the inventionspecifically or selectively recognize one or more epitopes of FcγRIIB,particularly native human FcγRIIB Another embodiment of the inventionencompasses the use of phage display technology to increase the affinityof the antibodies of the invention for FcγRIIB. Any screening methodknown in the art can be used to identify mutant antibodies withincreased avidity for FcγRIIB (e.g., ELISA). In another specificembodiment, antibodies of the invention are screened using antibodyscreening assays well known in the art (e.g., BIACORE assays) toidentify antibodies with K_(off) rate less than 3×10⁻³ s⁻¹.

Hybridoma clone 8B5.3.4s was deposited with the American Type CultureCollection (“ATCC”), having an address of 10801 University Blvd.,Manassas, Va., 20110-2209, on May 23, 2006 under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure and the Regulationsthereunder (Budapest Treaty); the deposited clone was found to be viableand was accorded ATCC accession number PTA-7610. In a preferredembodiment, the invention provides a monoclonal antibody produced byhybridoma clone 8B5.3.4, having ATCC accession number PTA-7610, orchimeric, humanized or other engineered versions thereof. In anotherembodiment, the invention provides a monoclonal antibody produced byclones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, and 1F2 having ATCC Accessionnumbers PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, andPTA-5959, respectively, or chimeric, humanized or other engineeredversions thereof. In another embodiment, the invention provides anisolated antibody or a fragment thereof that competes for binding withthe monoclonal antibody produced by clone 8B5.3.4 and binds FcγRIIB,preferably native human FcγRIIB with a greater affinity than saidantibody or a fragment thereof binds FcγRIIA, preferably native humanFcγRIIA and/or binds to the same epitope of FcγRIIB as the monoclonalantibody produced from clone 8B5.3.4 and binds FcγRIIB with a greateraffinity than said antibody or a fragment thereof binds FcγRIIA.Furthermore, the invention provides hybridoma cell line 8B5.3.4, 2B6,3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 having ATCC accession numbers PTA-7610,PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, andPTA-5959, respectively. In one specific embodiment, the inventionprovides the use of a 8B5.3.4, 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2antibody, or chimeric, humanized or other engineered versions thereof,to prevent, treat, manage or ameliorate a B-cell malignancy, or one ormore symptoms thereof. In one particular embodiment, an engineeredversion comprises one or more mutations in the Fc region. The one ormore mutations in the Fc region may result in an antibody with analtered antibody-mediated effector function, an altered binding to otherFc receptors (e.g., Fc activation receptors), an altered ADCC activity,or an altered C1q binding activity, or an altered complement dependentcytotoxicity activity, or any combination thereof. In a preferredembodiment, a humanized 8B5.3.4 antibody comprises a heavy chainvariable domain having the amino acid sequence of SEQ ID NO: 4 and alight chain variable domain having the amino acid sequence of SEQ ID NO:3. In another preferred embodiment, the Fc domain of the heavy chain ofthe humanized 8B5.3.4 antibody is engineered to comprise at least oneamino acid substitution at position 240, 243, 247, 255, 270, 292, 300,316, 370, 392, 396, 416, 419, or 421 with another amino acid at thatposition. In a more preferred embodiment, the Fc domain of the heavychain of the humanized 8B5.3.4 antibody has a leucine at position 247, alysine at position 421 and a glutamic acid at position 270; a threonineat position 392, a leucine at position 396, and a glutamic acid atposition 270; or a glutamic acid at position 270, an aspartic acid atposition 316, and a glycine at position 416. In certain embodiments ofthe invention, the antibody is not a monoclonal antibody produced byhybridoma clone 8B5.3.4, or chimeric, humanized or other engineeredversions thereof.

In certain embodiments of the invention, humanized 8B5.3.4 antibodiesare provided, said humanized 8B5.3.4 antibodies comprising a heavy chainvariable domain having the amino acid sequence of SEQ ID NO: 4 and alight chain variable domain having the amino acid sequence of SEQ ID NO:3, wherein the Fc domain of the heavy chain of the humanized 8B5.3.4antibody has a leucine at position 247, a lysine at position 421 and aglutamic acid at position 270; or a glutamic acid at position 270, anaspartic acid at position 316, and a glycine at position 416.

The invention also encompass polynucleotides that encode the antibodiesof the invention. In one embodiment, the invention provides an isolatednucleic acid sequence encoding a heavy chain or a light chain of anantibody or a fragment thereof that specifically binds FcγRIIB withgreater affinity than said antibody or a fragment thereof binds FcγRIIA.The invention also relates to a vector comprising said nucleic acid. Theinvention further provides a vector comprising a first nucleic acidmolecule encoding a heavy chain and a second nucleic acid moleculeencoding a light chain, said heavy chain and light chain being of anantibody or a fragment thereof that specifically binds FcγRIIB withgreater affinity than said antibody or a fragment thereof binds FcγRIIA.In one specific embodiment, said vector is an expression vector. Theinvention further provides host cells containing the vectors of orpolynucleotides encoding the antibodies of the invention. Preferably,the invention encompasses polynucleotides encoding heavy and lightchains of the antibodies produced by the deposited hybridoma clone8B5.3.4, 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 having ATCC accessionnumbers PTA-7610, PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively, or portions thereof, e.g., CDRs,variable domains, etc. and humanized versions thereof.

Activating and inhibitory Fc receptors, e.g., FcγRIIA and FcγRIIB, arecritical for the balanced function of these receptors and propercellular immune responses. The invention encompasses the use of theantibodies of the invention for the treatment of any disease related toloss of such balance and regulated control in the Fc receptor signalingpathway. Thus, the FcγRIIB antibodies of the invention have uses inregulating the immune response, e.g., in inhibiting immune response inconnection with autoimmune or inflammatory disease, or allergicresponse. The FcγRIIB antibodies of the invention can also be used toalter certain effector functions to enhance, for example, therapeuticantibody-mediated cytotoxicity.

The antibodies of the invention are useful for prevention or treatmentof cancer, for example, in one embodiment, as a single agent therapy. Ina preferred embodiment, the antibodies of the invention are used for thetreatment and/or prevention of melanoma. In another embodiment, theantibodies are useful for prevention or treatment of cancer,particularly in potentiating the cytotoxic activity of cancerantigen-specific therapeutic antibodies with cytotoxic activity toenhance tumor cell killing and/or enhancing antibody-dependentcell-mediated cytotoxicity (“ADCC”), complement-dependent cytotoxicity(“CDC”), or phagocytosis of the therapeutic antibodies. The inventionprovides a method of treating cancer in a patient having a cancercharacterized by a cancer antigen, said method comprising administeringto said patient a therapeutically effective amount of a first antibodyor a fragment thereof that specifically binds FcγRIIB with greateraffinity than said antibody or a fragment thereof binds FcγRIIA, and asecond antibody that specifically binds said cancer antigen and iscytotoxic. The invention also provides a method of treating cancer in apatient having a cancer characterized by a cancer antigen, said methodcomprising administering to said patient a therapeutically effectiveamount of an antibody or a fragment thereof that specifically bindsFcγRIIB, particularly native human FcγRIIB with greater affinity thansaid antibody or a fragment thereof binds FcγRIIA, preferably nativehuman FcγRIIA, and the constant domain of which further has an increasedaffinity for one or more Fc activation receptors, such as FcγRIIIA, whenthe antibody is monomeric, and an antibody that specifically binds saidcancer antigen and is cytotoxic. In one particular embodiment, said Fcactivation receptor is FcγRIIIA In particular embodiments, the antibodyof the invention is administered at a dose such that the antibody doesnot detectably bind to neutrophils.

In another preferred embodiment of the invention, the antibodies of theinvention are useful for prevention or treatment of B-cell malignancies,particularly non-Hodgkin's lymphoma or chronic lymphocytic leukemia.Accordingly, the present invention provides methods of treating,managing, preventing, or ameliorating a B-cell malignancy byadministering, either alone or in combination with one or more othertherapeutics, antibodies that specifically bind FcγRIIB, and,preferably, do not specifically bind FcγRIIA, as well as derivatives,analogs and antigen binding fragments of such antibodies. In particularembodiments, the cancer of the subject is refractory to one or morestandard or experimental therapies, particularly, to Rituxan treatment.The methods of the invention may be used for the treatment, management,prevention, or amelioration of B-cell diseases, such as, B-cell chroniclymphocytic leukemia (B-CLL), non-Hodgkin's lymphoma, diffuse large Bcell lymphoma, follicular lymphoma with areas of diffuse large B celllymphoma, small lymphocytic lymphoma, mantle cell lymphoma, and diffusesmall cleaved cell lymphoma.

In another embodiment, the invention provides for the use of aFcγRIIB-specific antibody conjugated to a therapeutic agent or drug.Examples of therapeutic agents which may be conjugated to ananti-FcγRIIB antibody or an antigen-binding fragment thereof include,but are not limited to, cytokines, toxins, radioactive elements, andantimetabolites.

In one embodiment, the invention provides for the use of anFcγRIIB-specific antibody in combination with a standard or experimentaltreatment regimen for B-cell malignancies (e.g., chemotherapy,radioimmunotherapy, or radiotherapy). Such combination therapy mayenhance the efficacy of standard or experimental treatment. Examples oftherapeutic agents that are particularly useful in combination with aFcγRIIB-specific antibody or an antigen-binding fragment thereof, forthe prevention, treatment, management, or amelioration of B-cellmalignancies, include, but are not limited to, Rituxan,interferon-alpha, and anti-cancer agents. Chemotherapeutic agents thatcan be used in combination with a FcγRIIB-specific antibody or anantigen-binding fragment thereof, include, but are not limited toalkylating agents, antimetabolites, natural products, and hormones. Thecombination therapies of the invention enable lower dosages of ananti-FcγRIIB antibody or an antigen-binding fragment thereof and/or lessfrequent administration of anti-FcγRIIB antibody or an antigen-bindingfragment thereof to a subject with a B-cell malignancy, to achieve atherapeutic or prophylactic effect.

In another embodiment, the use of an anti-FcγRIIB antibody or anantigen-binding fragment thereof prolongs the survival of a subjectdiagnosed with a B-cell malignancy.

In another embodiment, the invention provides a method of enhancing anantibody mediated cytotoxic effect in a subject being treated with acytotoxic antibody, said method comprising administering to said patientan antibody of the invention or a fragment thereof, in an amountsufficient to enhance the cytotoxic effect of said cytotoxic antibody.In yet another embodiment, the invention provides a method of enhancingan antibody-mediated cytotoxic effect in a subject being treated with acytotoxic antibody, said method comprising administering to said patientan antibody of the invention or a fragment thereof, further having anenhanced affinity for an Fc activation receptor, when monomeric, in anamount sufficient to enhance the cytotoxic effect of said cytotoxicantibody. In yet another embodiment, the invention provides a methodfurther comprising the administration of one or more additional cancertherapies.

The invention encompasses the use of the antibodies of the invention incombination with any therapeutic antibody that mediates its therapeuticeffect through cell killing to potentiate the antibody's therapeuticactivity. In one particular embodiment, the antibodies of the inventionpotentiate the antibody's therapeutic activity by enhancingantibody-mediated effector function. In another embodiment of theinvention, the antibodies of the invention potentiate the cytotoxicantibody's therapeutic activity by enhancing phagocytosis andopsonization of the targeted tumor cells. In yet another embodiment ofthe invention, the antibodies of the invention potentiate the antibody'stherapeutic activity by enhancing antibody-dependent cell-mediatedcytotoxicity (“ADCC”) in destruction of the targeted tumor cells. Incertain embodiments, the antibodies of the invention are used incombination with Fc fusion proteins to enhance ADCC.

In some embodiments, the invention encompasses use of the antibodies ofthe invention in combination with a therapeutic antibody that does notmediate its therapeutic effect through cell killing to potentiate theantibody's therapeutic activity. In a specific embodiment, the inventionencompasses use of the antibodies of the invention in combination with atherapeutic apoptosis-inducing antibody with agonistic activity, e.g.,anti-Fas antibody. Therapeutic apoptosis-inducing antibodies may bespecific for any death receptor known in the art for the modulation ofapoptotic pathway, e.g., TNFR receptor family member or a TRAIL familymember.

The invention encompasses using the antibodies of the invention to blockmacrophage mediated tumor cell progression and metastasis. Theantibodies of the invention are particularly useful in the treatment ofsolid tumors, where macrophage infiltration occurs. The antagonisticantibodies of the invention are particularly useful for controlling,e.g., reducing or eliminating, tumor cell metastasis, by reducing oreliminating the population of macrophages that are localized at thetumor site. The invention further encompasses antibodies thateffectively deplete or eliminate immune effector cells other thanmacrophages that express FcγRIIB, e.g., dendritic cells. Effectivedepletion or elimination of immune effector cells using the antibodiesof the invention may range from a reduction in population of theeffector cells by 50%, 60%, 70%, 80%, preferably 90%, and mostpreferably 99%. In particular embodiments, the antibody of the inventionis administered at a dose such that the antibody does not detectablybind to neutrophils.

In some embodiments, the agonistic antibodies of the invention areparticularly useful for the treatment of tumors of non-hematopoieticorigin, including tumors of melanoma cells.

In some embodiments, the invention encompasses use of the antibodies ofthe invention in combination with therapeutic antibodies thatimmunospecifically bind to tumor antigens that are not expressed on thetumor cells themselves, but rather on the surrounding reactive and tumorsupporting, non-malignant cells comprising the tumor stroma. In apreferred embodiment, an antibody of the invention is used incombination with an antibody that immunospecifically binds a tumorantigen on a fibroblast cell, e.g., fibroblast activation protein (FAP).

The invention provides a method of treating an autoimmune disorder in apatient in need thereof, said method comprising administering to saidpatient a therapeutically effective amount of one or more antibodies ofthe invention. The invention also provides a method of treating anautoimmune disorder in a patient in need thereof, said method furthercomprising administering to said patient a therapeutically effectiveamount of one or more anti-inflammatory agents, and/or one or moreimmunomodulatory agents.

The invention also provides a method of treating an inflammatorydisorder in a patient in need thereof, said method comprisingadministering to said patient a therapeutically effective amount of oneor more antibodies of the invention. The invention also provides amethod of treating an inflammatory disorder in a patient in needthereof, said method further comprising administering to said patient atherapeutically effective amount of one or more anti-inflammatoryagents, and/or one or more immunomodulatory agents.

The invention provides a method of enhancing an immune response to avaccine composition in a subject, said method comprising administeringto said subject an antibody or an antigen-binding fragment thereof thatspecifically binds FcγRIIB with greater affinity than said antibody or afragment thereof binds FcγRIIA, and a vaccine composition, such thatsaid antibody or a fragment thereof is administered in an amounteffective to enhance the immune response to said vaccine composition insaid subject. The antibodies of the invention may be used to enhance ahumoral and/or cell mediated response against the antigen(s) of thevaccine composition. The antibodies of the invention may be used incombination with any vaccines known in the art. The inventionencompasses the use of the antibodies of the invention to either preventor treat a particular disorder, where an enhanced immune responseagainst a particular antigen or antigens is effective to treat orprevent the disease or disorder.

The invention further provides a method for treating or preventing anIgE-mediated allergic disorder in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of theagonistic antibodies of the invention. The invention also provides amethod for treating or preventing an IgE-mediated allergic disorder in apatient in need thereof, comprising administering to said patient theantibodies of the invention in combination with other therapeuticantibodies or vaccine compositions used for the treatment or preventionof IgE-mediated allergic disorders.

The invention also provides a method for enhancing immune therapy for aninfectious agent wherein the antibodies of the invention areadministered to a patient that is already infected by a pathogen, suchas HIV, HCV or HSV, to enhance opsonization and phagocytosis of infectedcells.

The invention provides a method of treating diseases with impairedapoptotic mediated signaling, e.g., cancer, autoimmune disease. In aspecific embodiment, the invention encompasses a method of treating adisease with deficient Fas-mediated apoptosis, said method comprisingadministering an antibody of the invention in combination with ananti-Fas antibody.

The invention encompasses the use of the antibodies of the invention todetect the presence of FcγRIIB specifically (i.e., FcγRIIB and notFcγRIIA) in a biological sample.

In another embodiment, the invention provides a method of diagnosis ofan autoimmune disease in a subject comprising: (i) contacting abiological sample from said subject with an effective amount of anantibody of the invention; and (ii) detecting binding of said antibodyor a fragment thereof, wherein detection of said detectable marker abovea background or standard level indicates that said subject has anautoimmune disease.

The invention further provides a pharmaceutical composition comprising(i) a therapeutically effective amount of the antibody or a fragmentthereof that specifically binds FcγRIIB with greater affinity than saidantibody or a fragment thereof binds FcγRIIA; and (ii) apharmaceutically acceptable carrier. The invention additionally providesa pharmaceutical composition comprising (i) a therapeutically effectiveamount of the antibody or fragment thereof that specifically bindsFcγRIIB with greater affinity than said antibody or fragment thereofbinds FcγRIIA; (ii) a cytotoxic antibody that specifically binds acancer antigen; and (iii) a pharmaceutically acceptable carrier.

In certain embodiments of the invention, pharmaceutical compositions areprovided for use in accordance with the methods of the invention, saidpharmaceutical compositions comprising an anti-FcγRIIB antibody or anantigen-binding fragment thereof, in an amount effective to prevent,treat, manage, or ameliorate a B-cell malignancy, or one or moresymptoms thereof, and a pharmaceutically acceptable carrier. Theinvention also provides pharmaceutical compositions for use inaccordance with the methods of the invention, said pharmaceuticalcompositions comprising an anti-FcγRIIB antibody or an antigen-bindingfragment thereof, a prophylactic or therapeutic agent other than aFcγRIIB antagonist, and a pharmaceutically acceptable carrier.

3.1 Definitions

As used herein, the term “specifically binds to FcγRIIB” and analogousterms refer to antibodies or fragments thereof (or any other FcγRIIBbinding molecules) that specifically bind to FcγRIIB or a fragmentthereof and do not specifically bind to other Fc receptors, inparticular to FcγRIIA. Further it is understood to one skilled in theart, that an antibody that specifically binds to FcγRIIB, may bindthrough the variable domain or the constant domain of the antibody. Ifthe antibody that specifically binds to FcγRIIB binds through itsvariable domain, it is understood to one skilled in the art that it isnot aggregated, i.e., is monomeric. An antibody that specifically bindsto FcγRIIB may bind to other peptides or polypeptides with loweraffinity as determined by, e.g., immunoassays, BIAcore, or other assaysknown in the art. Preferably, antibodies or fragments that specificallybind to FcγRIIB or a fragment thereof do not cross-react with otherantigens. Antibodies or fragments that specifically bind to FcγRIIB canbe identified, for example, by immunoassays, BIAcore, or othertechniques known to those of skill in the art. An antibody or a fragmentthereof binds specifically to a FcγRIIB when it binds to FcγRIIB withhigher affinity than to any cross-reactive antigen as determined usingexperimental techniques, such as western blots, radioimmunoassays (RIA)and enzyme-linked immunosorbent assays (ELISAs). See, e.g., Paul, ed.,1989, Fundamental Immunology Second Edition, Raven Press, New York atpages 332-336 for a discussion regarding antibody specificity.

As used herein, the term “native FcγRIIB” refers to FcγRIIB which isendogenously expressed and present on the surface of a cell. In someembodiments, “native FcγRIIB” encompasses a protein that isrecombinantly expressed in a mammalian cell. Preferably, the nativeFcγRIIB is not expressed in a bacterial cell, i.e., E. coli. Mostpreferably the native FcγRIIB is not denatured, i.e., it is in itsbiologically active conformation.

As used herein, the term “native FcγRIIA” refers to FcγRIIA which isendogenously expressed and present on the surface of a cell. In someembodiments, “native FcγRIIA” encompasses a protein that isrecombinantly expressed in a mammalian cell. Preferably, the nativeFcγRIIA is not expressed in a bacterial cell, i.e., E. coli. Mostpreferably the native FcγRIIA is not denatured, i.e., it is in itsbiologically active conformation.

As used herein, the term “endogenous” in the context of a cellularprotein refers to protein naturally occurring and/or expressed by thecell in the absence of recombinant manipulation; accordingly, the terms“endogenously expressed protein” or “endogenous protein” excludescellular proteins expressed by means of recombinant technology.

As used herein, the term “analog” in the context of proteinaceous agents(e.g., proteins, polypeptides, and antibodies) refers to a proteinaceousagent that possesses a similar or identical function as a secondproteinaceous agent but does not necessarily comprise a similar oridentical amino acid sequence of the second proteinaceous agent, orpossess a similar or identical structure of the second proteinaceousagent. A proteinaceous agent that has a similar amino acid sequencerefers to a second proteinaceous agent that satisfies at least one ofthe following: (a) a proteinaceous agent having an amino acid sequencethat is at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least99% identical to the amino acid sequence of a second proteinaceousagent; (b) a proteinaceous agent encoded by a nucleotide sequence thathybridizes under stringent conditions to a nucleotide sequence encodinga second proteinaceous agent of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least 80 contiguous amino acid residues, atleast 90 contiguous amino acid residues, at least 100 contiguous aminoacid residues, at least 125 contiguous amino acid residues, or at least150 contiguous amino acid residues; and (c) a proteinaceous agentencoded by a nucleotide sequence that is at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% identical to the nucleotidesequence encoding a second proteinaceous agent. A proteinaceous agentwith similar structure to a second proteinaceous agent refers to aproteinaceous agent that has a similar secondary, tertiary or quaternarystructure to the second proteinaceous agent. The structure of apolypeptide can be determined by methods known to those skilled in theart, including but not limited to, peptide sequencing, X-raycrystallography, nuclear magnetic resonance, circular dichroism, andcrystallographic electron microscopy.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul,1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.,1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performedwith the NBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the present invention. BLAST protein searches can beperformed with the XBLAST program parameters set, e.g., to score-50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,the NCBI website). Another preferred, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

As used herein, the term “analog” in the context of a non-proteinaceousagent refers to a second organic or inorganic molecule which possess asimilar or identical function as a first organic or inorganic moleculeand is structurally similar to the first organic or inorganic molecule.

As used herein, the terms “antagonist” and “antagonists” refer to anyprotein, polypeptide, peptide, antibody, antibody fragment, largemolecule, or small molecule (less than 10 kD) that blocks, inhibits,reduces or neutralizes a function, activity and/or expression of anothermolecule, such as that of FcγRIIB In various embodiments, an antagonistreduces a function, activity and/or expression of another molecule by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 99% relative to acontrol such as phosphate buffered saline (PBS).

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,camelized antibodies, single-chain Fvs (scFv), single chain antibodies,Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv),intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id and anti-anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

As used herein, the terms “B-cell malignancies” and “B-cell malignancy”refer to any B-cell lymphoproliferative disorder. B-cell malignanciesinclude tumors of B-cell origin. B-cell malignancies include, but arenot limited to, lymphomas, chronic lymphocytic leukemias, acutelymphoblastic leukemias, multiple myeloma, Hodgkin's and non-Hodgkin'sdisease, diffuse large B cell lymphoma, follicular lymphoma with areasof diffuse large B cell lymphoma, small lymphocytic lymphoma, mantlecell lymphoma, and diffuse small cleaved cell lymphoma.

Unless otherwise indicated, when referring to antibodies (as broadlydefined herein), reference to antibody domains and/or amino acidpositions within antibodies, or fragments thereof, is in accordance withthe definition and assignment of amino acids to each domain in Kabat etal, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5^(th) Ed. PublicHealth Service (National Institutes of Health, Bethesda, Md., 1987 and1991); incorporated herein by reference in its entirety. Amino acidsfrom the variable regions of the mature heavy and light chains ofimmunoglobulins are designated by the position of an amino acid in thechain. Kabat described numerous amino acid sequences for antibodies,identified an amino acid consensus sequence for each subgroup, andassigned a residue number to each amino acid. Kabat's numbering schemeis extendible to antibodies not included in his compendium by aligningthe antibody in question with one of the consensus sequences in Kabat byreference to conserved amino acids. This method for assigning residuenumbers has become standard in the field and readily identifies aminoacids at equivalent positions in different antibodies, includingchimeric or humanized variants. For example, an amino acid at position50 of a human antibody light chain occupies the equivalent position toan amino acid at position 50 of a mouse antibody light chain. Thus, asused herein in the context of humanized antibodies, a reference such as“at position 297 of the Fc region” refers to the amino acid position inan immunoglobulin chain, region of an a immunoglobulin chain, or regionof a polypeptide derived from an immunoglobulin chain, that correspondsto position 297 of the corresponding human immunoglobulin.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes, leukemias and lymphomas. The term “cancer”refers to a disease involving cells that have the potential tometastasize to distal sites and exhibit phenotypic traits that differfrom those of non-cancer cells, for example, formation of colonies in athree-dimensional substrate such as soft agar or the formation oftubular networks or weblike matrices in a three-dimensional basementmembrane or extracellular matrix preparation. Non-cancer cells do notform colonies in soft agar and form distinct sphere-like structures inthree-dimensional basement membrane or extracellular matrixpreparations. Cancer cells acquire a characteristic set of functionalcapabilities during their development, albeit through variousmechanisms. Such capabilities include evading apoptosis,self-sufficiency in growth signals, insensitivity to anti-growthsignals, tissue invasion/metastasis, limitless explicative potential,and sustained angiogenesis. The term “cancer cell” is meant to encompassboth pre-malignant and malignant cancer cells. In some embodiments,cancer refers to a benign tumor, which has remained localized. In otherembodiments, cancer refers to a malignant tumor, which has invaded anddestroyed neighboring body structures and spread to distant sites. Inyet other embodiments, the cancer is associated with a specific cancerantigen.

As used herein, the term “derivative” in the context of polypeptides orproteins, including antibodies, refers to a polypeptide or protein thatcomprises an amino acid sequence which has been altered by theintroduction of amino acid residue substitutions, deletions oradditions. The term “derivative” as used herein also refers to apolypeptide or protein which has been modified, i.e., by the covalentattachment of any type of molecule to the polypeptide or protein. Forexample, but not by way of limitation, an antibody may be modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. A derivative polypeptide or protein may be produced by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Further, aderivative polypeptide or protein derivative possesses a similar oridentical function as the polypeptide or protein from which it wasderived.

The term “derivative” as used herein in conjunction with FcγRIIB refersto a polypeptide that comprises an amino acid sequence of a FcγRIIBpolypeptide, a fragment of a FcγRIIB polypeptide, an antibody thatimmunospecifically binds to a FcγRIIB polypeptide, or an antibodyfragment that immunospecifically binds to a FcγRIIB polypeptide, thathas been altered by the introduction of amino acid residuesubstitutions, deletions or additions (i.e., mutations). In someembodiments, an antibody derivative or fragment thereof comprises aminoacid residue substitutions, deletions or additions in one or more CDRs.The antibody derivative may have substantially the same binding, betterbinding, or worse binding when compared to a non-derivative antibody. Inspecific embodiments, one, two, three, four, or five amino acid residuesof the CDR have been substituted, deleted or added (i.e., mutated). Theterm “derivative” as used herein in conjunction with FcγRIIB also refersto a FcγRIIB polypeptide, a fragment of a FcγRIIB polypeptide, anantibody that immunospecifically binds to a FcγRIIB polypeptide, or anantibody fragment that immunospecifically binds to a FcγRIIB polypeptidewhich has been modified, i.e., by the covalent attachment of any type ofmolecule to the polypeptide. For example, but not by way of limitation,a FcγRIIB polypeptide, a fragment of a FcγRIIB polypeptide, an antibody,or antibody fragment may be modified, e.g., by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. A derivative of a FcγRIIBpolypeptide, a fragment of a FcγRIIB polypeptide, an antibody, orantibody fragment may be modified by chemical modifications usingtechniques known to those of skill in the art, including, but notlimited to, specific chemical cleavage, acetylation, formulation,metabolic synthesis of tunicamycin, etc. Further, a derivative of aFcγRIIB polypeptide, a fragment of a FcγRIIB polypeptide, an antibody,or antibody fragment may contain one or more non-classical amino acids.In one embodiment, an antibody derivative possesses a similar oridentical function as the parent antibody. In another embodiment, aderivative of an antibody, or antibody fragment has an altered activitywhen compared to an unaltered antibody. For example, a derivativeantibody or fragment thereof can bind to its epitope more tightly or bemore resistant to proteolysis.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

As used herein, the term “epitope” refers to a region on an antigenmolecule to which an antibody specifically binds.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide. Preferably, antibodyfragments are epitope binding fragments.

As used herein, the term “humanized antibody” refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody, because, e.g., the entire variable region ofa chimeric antibody is non-human. One says that the donor antibody hasbeen “humanized”, by the process of “humanization”, because theresultant humanized antibody is expected to bind to the same antigen asthe donor antibody that provides the CDR's. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichhypervariable region residues of the recipient are replaced byhypervariable region residues from a non-human species (donor antibody)such as mouse, rat, rabbit or non-human primate having the desiredspecificity, affinity, and capacity. In some instances, Framework Region(FR) residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin that immunospecifically binds to a FcγRIIBpolypeptide, that has been altered by the introduction of amino acidresidue substitutions, deletions or additions (i.e., mutations). In someembodiments, a humanized antibody is a derivative. Such a humanizedantibody comprises amino acid residue substitutions, deletions oradditions in one or more non-human CDRs. The humanized antibodyderivative may have substantially the same binding, better binding, orworse binding when compared to a non-derivative humanized antibody. Inspecific embodiments, one, two, three, four, or five amino acid residuesof the CDR have been substituted, deleted or added (i.e., mutated). Forfurther details in humanizing antibodies, see European Patent Nos. EP239,400, EP 592,106, and EP 519,596; International Publication Nos. WO91/09967 and WO 93/17105; U.S. Pat. Nos. 5,225,539, 5,530,101,5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991,Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, ProteinEngineering 7(6):805-814; Roguska et al., 1994, Proc Natl Acad Sci USA91:969-973; Tan et al., 2002, J. Immunol. 169:1119-25; Caldas et al.,2000, Protein Eng. 13:353-60; Morea et al., 2000, Methods 20:267-79;Baca et al., 1997, J. Biol. Chem. 272:10678-84; Roguska et al., 1996,Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55 (23Supp):5973s-5977s; Couto et al., 1995, Cancer Res. 55:1717-22; Sandhu,1994, Gene 150:409-10; Pedersen et al., 1994, J. Mol. Biol. 235:959-73;Jones et al., 1986, Nature 321:522-525; Reichmann et al., 1988, Nature332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody which are responsible for antigen binding. Thehypervariable region comprises amino acid residues from a“Complementarity Determining Region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (i.e.,residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, 1987, J. Mol. Biol.196:901-917). CDR residues for Eph099B-208.261 and Eph099B-233.152 arelisted in Table 1. “Framework Region” or “FR” residues are thosevariable domain residues other than the hypervariable region residues asherein defined.

As used herein, the term “immunomodulatory agent” and variations thereofincluding, but not limited to, immunomodulatory agents, refer to anagent that modulates a host's immune system. In certain embodiments, animmunomodulatory agent is an immunosuppressant agent. In certain otherembodiments, an immunomodulatory agent is an immunostimulatory agent.Immunomodulatory agents include, but are not limited to, smallmolecules, peptides, polypeptides, fusion proteins, antibodies,inorganic molecules, mimetic agents, and organic molecules.

As used herein, the terms “manage,” “managing” and “management” refer tothe beneficial effects that a subject derives from administration of aprophylactic or therapeutic agent, which does not result in a cure ofthe disease. In certain embodiments, a subject is administered one ormore prophylactic or therapeutic agents to “manage” a disease so as toprevent the progression or worsening of the disease.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the occurrence and/or recurrence or onset of one ormore symptoms of a disorder in a subject resulting from theadministration of a prophylactic or therapeutic agent.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder. A prophylacticallyeffective amount may refer to the amount of prophylactic agentsufficient to prevent the recurrence or spread of hyperproliferativedisease, particularly cancer, or the occurrence of such in a patient,including but not limited to those predisposed to hyperproliferativedisease, for example those genetically predisposed to cancer orpreviously exposed to carcinogens. A prophylactically effective amountmay also refer to the amount of the prophylactic agent that provides aprophylactic benefit in the prevention of disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of disease. Used in connection with an amount of anFcγRIIB antibody of the invention, the term can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy of orsynergies with another prophylactic agent, such as but not limited to atherapeutic antibody. In certain embodiments, the term “prophylacticagent” refers to an agonistic FcγRIIB-specific antibody. In otherembodiments, the term “prophylactic agent” refers to an antagonisticFcγRIIB-specific antibody. In certain other embodiments, the term“prophylactic agent” refers to cancer chemotherapeutics, radiationtherapy, hormonal therapy, biological therapy (e.g., immunotherapy),and/or FcγRIIB antibodies of the invention. In other embodiments, morethan one prophylactic agent may be administered in combination.

As used herein, the phrase “side effects” encompasses unwanted andadverse effects of a prophylactic or therapeutic agent. Adverse effectsare always unwanted, but unwanted effects are not necessarily adverse.An adverse effect from a prophylactic or therapeutic agent might beharmful or uncomfortable or risky. Side effects from chemotherapyinclude, but are not limited to, gastrointestinal toxicity such as, butnot limited to, early and late-forming diarrhea and flatulence, nausea,vomiting, anorexia, leukopenia, anemia, neutropenia, asthenia, abdominalcramping, fever, pain, loss of body weight, dehydration, alopecia,dyspnea, insomnia, dizziness, mucositis, xerostomia, and kidney failure,as well as constipation, nerve and muscle effects, temporary orpermanent damage to kidneys and bladder, flu-like symptoms, fluidretention, and temporary or permanent infertility. Side effects fromradiation therapy include but are not limited to fatigue, dry mouth, andloss of appetite. Side effects from biological therapies/immunotherapiesinclude but are not limited to rashes or swellings at the site ofadministration, flu-like symptoms such as fever, chills and fatigue,digestive tract problems and allergic reactions. Side effects fromhormonal therapies include but are not limited to nausea, fertilityproblems, depression, loss of appetite, eye problems, headache, andweight fluctuation. Additional undesired effects typically experiencedby patients are numerous and known in the art, see, e.g., thePhysicians' Desk Reference (56^(th) ed., 2002), which is incorporatedherein by reference in its entirety.

As used herein, the terms “single-chain Fv” or “scFv” refer to antibodyfragments comprise the VH and VL domains of antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. For a review of sFv see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994). In specific embodiments,scFvs include bi-specific scFvs and humanized scFvs.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey and human), most preferably a human.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder associated with FcγRIIB and any disease related to the lossof regulation in the Fc receptor signaling pathway or to enhance thetherapeutic efficacy of another therapy, e.g., therapeutic antibody,vaccine therapy or prophylaxis, etc. A therapeutically effective amountmay refer to the amount of therapeutic agent sufficient to delay orminimize the onset of disease, e.g., delay or minimize the spread ofcancer. A therapeutically effective amount may also refer to the amountof the therapeutic agent that provides a therapeutic benefit in thetreatment or management of a disease. Further, a therapeuticallyeffective amount with respect to a therapeutic agent of the inventionmeans that amount of therapeutic agent alone, or in combination withother therapies, that provides a therapeutic benefit in the treatment ormanagement of a disease, e.g., sufficient to enhance the therapeuticefficacy of a therapeutic antibody sufficient to treat or manage adisease. Used in connection with an amount of FcγRIIB antibody of theinvention, the term can encompass an amount that improves overalltherapy, reduces or avoids unwanted effects, or enhances the therapeuticefficacy of or synergies with another therapeutic agent.

As used herein, the terms “treat,” “treating” and “treatment” refer tothe eradication, reduction or amelioration of symptoms of a disease ordisorder related to the loss of regulation in the Fc receptor signalingpathway or to enhance the therapeutic efficacy of another therapy, e.g.,a therapeutic antibody, vaccine therapy or prophylaxis. In someembodiments, treatment refers to the eradication, removal, modification,or control of primary, regional, or metastatic cancer tissue thatresults from the administration of one or more therapeutic agents. Incertain embodiments, such terms refer to the minimizing or delaying thespread of cancer resulting from the administration of one or moretherapeutic agents to a subject with such a disease. In otherembodiments, such terms refer to elimination of disease causing cells.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder, e.g.,hyperproliferative cell disorder, especially cancer. A firstprophylactic or therapeutic agent can be administered prior to (e.g., 1minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours,4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 1 minute, 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after)the administration of a second prophylactic or therapeutic agent to asubject which had, has, or is susceptible to a disorder. Theprophylactic or therapeutic agents are administered to a subject in asequence and within a time interval such that the agent of the inventioncan act together with the other agent to provide an increased benefitthan if they were administered otherwise. Any additional prophylactic ortherapeutic agent can be administered in any order with the otheradditional prophylactic or therapeutic agents.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Immunoreactivity of 8B5.3.4 Antibody Against FcγRIIA and FcγRIIBThe direct binding of the antibody produced by the 8B5.3.4 hybridomacell line clone (MAb 8B5.3.4) to FcγRIIB and FcγRIIA was tested (induplicate) in an ELISA assay using plates coated with the receptorsFcγRIIA and FcγRIIB The bound antibodies were detected with a goatanti-mouse HRP conjugated antibody, and the absorbance was monitored at650 nm.

FIG. 2: Isotype Characterization of the MAb 8B5.3.4. The isotype of theMAb 8B5.3.4 produced by hybridoma clone 8B5.3.4 was determined by ELISAassay. Antibodies against the various isotypes were assayed, and theabsorbance values are reported.

FIG. 3: The nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 3)sequences for the variable light chain of the monoclonal antibodyproduced by clone 8B5.3.4 are represented.

FIG. 4: The nucleotide (SEQ ID NO: 2) and amino acid (SEQ ID NO: 4)sequences for the variable heavy chain of the monoclonal antibodyproduced by clone 8B5.3.4 are represented.

5. DESCRIPTION OF THE PREFERRED EMBODIMENTS 5.1 FcγRIIB-SpecificAntibodies

The present invention encompasses antibodies (preferably monoclonalantibodies) or fragments thereof that specifically bind FcγRIIB,preferably human FcγRIIB, more preferably native human FcγRIIB with agreater affinity than said antibodies or fragments thereof bind FcγRIIA,preferably human FcγRIIA, more preferably native human FcγRIIA.Representive antibodies are disclosed in U.S. Patent ApplicationPublication No. 2004/0185045, U.S. Provisional Application Ser. No.60/569,882, and U.S. patent application Ser. No. 11/126,978, hereinexpressly incorporated by reference in their entireties. The presentinvention encompasses the use of a FcγRIIB-specific antibody, an analog,derivative or an antigen-binding fragment thereof (e.g., one or morecomplementarity determining regions (“CDRs”) of a FcγRIIB-specificantibody) in the prevention, treatment, management or amelioration of adisease, such as cancer, in particular, a B-cell malignancy, or one ormore symptoms thereof. Preferably, the antibodies of the invention bindthe extracellular domain of native human FcγRIIB In certain embodiments,the antibodies or fragments thereof bind to FcγRIIB with an affinitygreater than two-fold, four fold, 6 fold, 10 fold, 20 fold, 50 fold, 100fold, 1000 fold, 10⁴ fold, 10⁵ fold, 10⁶ fold, 10⁷ fold, or 10⁸ foldthan said antibodies or fragments thereof bind FcγRIIA. In yet otherembodiments, the invention encompasses the use of FcγRIIB antibodiesthat bind exclusively to FcγRIIB and have no affinity for FcγRIIA usingstandard methods known in the art and disclosed herein. In a preferredembodiment, the antibodies are human or humanized.

In yet another preferred embodiment, the antibodies of the inventionfurther do not bind Fc activation receptors, e.g., FcγIIIA, FcγIIIB,etc. In one embodiment, the FcγRIIB-specific antibody in accordance withthe invention is not the monoclonal antibody designated KB61, asdisclosed in Pulford et al., 1986 (Immunology, 57: 71-76) or themonoclonal antibody designated MAbII8D2 as disclosed in Weinrich et al.,1996, (Hybridoma, 15(2):109-6). In a specific embodiment, theFcγRIIB-specific antibody of the invention does not bind to the sameepitope and/or does not compete with binding with the monoclonalantibody KB61 or II8D2. Preferably, the FcγRIIB-specific antibody of theinvention does not bind the amino acid sequence SDPNFSI corresponding topositions 135-141 of FcγRIIb2 isoform.

In a particular embodiment, the antibodies of the invention, orfragments thereof agonize at least one activity of FcγRIIB In oneembodiment of the invention, said activity is inhibition of B cellreceptor-mediated signaling. In another embodiment, the agonisticantibodies of the invention inhibit activation of B cells, B cellproliferation, antibody production, intracellular calcium influx of Bcells, cell cycle progression, or activity of one or more downstreamsignaling molecules in the FcγRIIB signal transduction pathway. In yetanother embodiment, the agonistic antibodies of the invention enhancephosphorylation of FcγRIIB or SHIP recruitment. In a further embodimentof the invention, the agonistic antibodies inhibit MAP kinase activityor Akt recruitment in the B cell receptor-mediated signaling pathway. Inanother embodiment, the agonistic antibodies of the invention agonizeFcγRIIB-mediated inhibition of FcεRI signaling. In a particularembodiment, said antibodies inhibit FcεRI-induced mast cell activation,calcium mobilization, degranulation, cytokine production, or serotoninrelease. In another embodiment, the agonistic antibodies of theinvention stimulate phosphorylation of FcγRIIB, stimulate recruitment ofSHIP, stimulate SHIP phosphorylation and its association with Shc, orinhibit activation of MAP kinase family members (e.g., Erk1, Erk2, JNK,p38, etc.). In yet another embodiment, the agonistic antibodies of theinvention enhance tyrosine phosphorylation of p62dok and its associationwith SHIP and rasGAP. In another embodiment, the agonistic antibodies ofthe invention inhibit FcγR-mediated phagocytosis in monocytes ormacrophages.

In another embodiment, the antibodies of the invention, or fragmentsthereof antagonize at least one activity of FcγRIIB In one embodiment,said activity is activation of B cell receptor-mediated signaling. In aparticular embodiment, the antagonistic antibodies of the inventionenhance B cell activity, B cell proliferation, antibody production,intracellular calcium influx, or activity of one or more downstreamsignaling molecules in the FcγRIIB signal transduction pathway. In yetanother particular embodiment, the antagonistic antibodies of theinvention decrease phosphorylation of FcγRIIB or SHIP recruitment. In afurther embodiment of the invention, the antagonistic antibodies enhanceMAP kinase activity or Akt recruitment in the B cell receptor mediatedsignaling pathway. In another embodiment, the antagonistic antibodies ofthe invention antagonize FcγRIIB-mediated inhibition of FcεRI signaling.In a particular embodiment, the antagonistic antibodies of the inventionenhance FcεRI-induced mast cell activation, calcium mobilization,degranulation, cytokine production, or serotonin release. In anotherembodiment, the antagonistic antibodies of the invention inhibitphosphorylation of FcγRIIB, inhibit recruitment of SHIP, inhibit SHIPphosphorylation and its association with Shc, enhance activation of MAPkinase family members (e.g., Erk1, Erk2, JNK, p38, etc.). In yet anotherembodiment, the antagonistic antibodies of the invention inhibittyrosine phosphorylation of p62dok and its association with SHIP andrasGAP. In another embodiment, the antagonistic antibodies of theinvention enhance FcγR-mediated phagocytosis in monocytes ormacrophages. In another embodiment, the antagonistic antibodies of theinvention prevent phagocytosis, clearance of opsonized particles bysplenic macrophages.

In other embodiments, the antibodies of the invention, or fragmentsthereof can be used to target one population of cells, but not others.Without being bound by any theory, the present inventors have discoveredthat FcγRIIB is not highly expressed on neutrophils, as previouslythought. High concentrations of an anti-FcγRIIB antibody react withneutrophils. However, neutrophil reactivity rapidly disappears withdecreasing concentrations of anti-FcγRIIB. At low concentrations ofanti-FcγRIIB antibody, reactivity with CD20+ B cells was preserved.Thus, reactivity of an antibody of the invention with neutrophils can bereduced so as to not affect irrelevant populations, such as neutrophilsor platelets. Accordingly, in certain embodiments of the invention, anantibody of the invention is employed at levels that fully recognize itstarget populations, but not other cells.

Antibodies of the invention include, but are not limited to, monoclonalantibodies, synthetic antibodies, recombinantly produced antibodies,multispecific antibodies, human antibodies, humanized antibodies,chimeric antibodies, camelized antibodies, single-chain Fvs (scFv),single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragmentsof any of the above. In particular, antibodies used in the methods ofthe present invention include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybind to FcγRIIB with greater affinity than said immunoglobulin moleculesbind FcγRIIA. Antibody analogs may also include FcγRIIB-specific T-cellreceptors, for example, chimeric T-cell receptors (see, e.g., U.S.Patent Application Publication No. 2004/0043401), a single-chain T-cellreceptor linked to a single-chain antibody (see, e.g., U.S. Pat. No.6,534,633), and protein scaffolds (see, e.g., U.S. Pat. No. 6,818,418).In certain embodiments, an antibody analog of the invention is not amonoclonal antibody.

The antibodies used in the methods of the invention may be from anyanimal origin including birds and mammals (e.g., human, non-humanprimate, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse,or chicken). Preferably, the antibodies are human or humanizedmonoclonal antibodies. As used herein, “human” antibodies includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries orlibraries of synthetic human immunoglobulin coding sequences or frommice that express antibodies from human genes.

The antibodies used in the methods of the present invention may bemonospecific, bispecific, trispecific or of greater multispecificity.Multispecific antibodies may immunospecifically bind to differentepitopes of FcγRIIB or immunospecifically bind to both an epitope ofFcγRIIB as well a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., InternationalPublication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793;Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893,4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al.,1992, J. Immunol. 148:1547-1553; Todorovska et al., 2001 Journal ofImmunological Methods, 248:47-66.

In particular embodiments, the antibodies of the invention aremulti-specific with specificities for FcγRIIB and for a cancer antigenor any other cell surface marker specific for a cell (e.g., an immunecell such as a T-cell or B-cell) designed to be killed, e.g., intreating or preventing a particular disease or disorder, or for other Fcreceptors, e.g., FcγRIIIA, FcγRIIIB, etc.

In one particular embodiment, the antibody is derived from a mousemonoclonal antibody produced by hybridoma clone, having ATCC accessionnumber PTA-7610. Hybridomas producing antibodies 8B5.3.4 have beendeposited with the American Type Culture Collection (10801 UniversityBlvd., Manassas, Va. 20110-2209) on May 23, 2006 under the provisions ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedures, and assignedaccession number PTA-7610 (for hybridoma producing the 8B5.3.4antibody), and are incorporated herein by reference. In a specificembodiment, the invention encompasses an antibody with the heavy chainvariable region having the amino acid sequence of SEQ ID NO: 4 and thelight chain variable region having the amino acid sequence of SEQ ID NO:3. In a preferred embodiment, the antibodies of the invention are humanor have been humanized, preferably a humanized version of the antibodyproduced by hybridoma clone 8B5.3.4.

The invention also encompasses the use of other antibodies, preferablymonoclonal antibodies or fragments thereof that specifically bindFcγRIIB, preferably human FcγRIIB, more preferably native human FcγRIIB,that are derived from clones including but not limited to 2B6 and 3H7,1D5, 2E1, 2H9, 2D11, and 1F2 having ATCC Accession numbers, PTA-4591,PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively. Hybridomas producing the 2B6 and 3H7 clones were depositedunder the provisions of the Budapest Treaty with the American TypeCulture Collection (10801 University Blvd., Manassas, Va. 20110-2209) onAug. 13, 2002, and are incorporated herein by reference. Hybridomasproducing the 1D5, 2E1, 2H9, 2D11, and 1F2 clones were deposited underthe provisions of the Budapest Treaty with the American Type CultureCollection (10801 University Blvd., Manassas, Va. 20110-2209) on May 7,2004, and are incorporated herein by reference. In preferredembodiments, the antibodies described above are chimerized or humanized.

In a specific embodiment, an antibody used in the methods of the presentinvention is an antibody or an antigen-binding fragment thereof (e.g.,comprising one or more complementarily determining regions (CDRs),preferably all 6 CDRs) of the antibody produced by clone 8B5.3.4 withATCC accession number PTA-7610 (e.g., the heavy chain CDR3). In aspecific embodiment, an antibody used in the methods of the presentinvention is an antibody or an antigen-binding fragment thereof (e.g.,comprising one or more complementarily determining regions (CDRs),preferably all 6 CDRs) of the antibody produced by clone 2B6, 3H7, 1D5,2E1, 2H9, 2D11, and 1F2 having ATCC Accession numbers, PTA-4591,PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively (e.g., the heavy chain CDR3). Antibodies or antigen-bindingfragments thereof comprising less than 6 CDRs with high affinity andspecificity for a particular antigen as well as methods for producingand identifying such antibodies are commonly known in the art (see,e.g., Ward et al., 1989, Nature 341:544-546; Dumoulin et al., 2002,Protein Sci. 11:500-515; Davies et al., 1995, Bio/Technol. 13:475-479;Van den Beucken et al., 2001, J. Mol. Biol. 310:591-601; and Pereira etal., 1998, Biochem. 37:1430-1437, all of which are incorporated byreference herein in their entireties). Antibodies specific for aparticular antigen that were generated by combining one or two CDRs froman antibody known to specifically bind to the antigen with other CDRshave been described and are commonly known in the art (see, e.g., Markset al., 1992, Bio/Technol. 10:779-783; Klimka et al., 2000, Brit. J.Cancer 83(2):252-260; and Rader et al., 1998, Proc. Natl. Acad. Sci. USA95:8910-8915, all of which are incorporated by reference herein in theirentireties). Thus, the invention contemplates antibodies having one,two, three, four, or five of the CDRs of clone 8B5.3.4 that bind FcγRIIBspecifically and which may be identified using the screening methodsdisclosed herein.

In one embodiment, an antibody fragment of the invention is apolypeptide comprising one more more CDRs of the antibody produced byclone 8B5.3.4 with ATCC accession number PTA-7610 (e.g., the heavy chainCDR3) that specifically binds the extracellular domain of native humanFcγRIIB with greater affinity than said polypeptide binds native humanFcγRIIA. The polypeptide of the invention may comprise an amino acidsequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, or any combination thereof (see, e.g., Davies etal., 1995, Bio/Technol. 13:475-479; Pereira et al., 1998, Biochemistry37:1430-1437; Tsumoto et al., 2002, FEBS Letters 525:77-82; van denBeucken et al., 2001, J. Mol. Biol. 310:591-601; Ward et al., 1989,Nature 341:544-546; and Dumoulin et al., 2002, Protein Sci. 11:500-515,all of which are incorporated by reference in their entireties).

In another embodiment, an antibody used in the methods of the presentinvention binds to the same epitope as the mouse monoclonal antibodyproduced by clone 8B5.3.4 with ATCC accession number PTA-7610,respectively and/or competes with the mouse monoclonal antibody producedby clone 8B5.3.4 with ATCC accession number PTA-7610, respectively asdetermined, e.g., in an ELISA assay or other appropriate competitiveimmunoassay, and also binds FcγRIIB with a greater affinity than saidantibody or a fragment thereof binds FcγRIIA. In another embodiment, anantibody used in the methods of the present invention binds to the sameepitope as the mouse monoclonal antibody produced by hybridoma clones2B6, 3H7, 1D5, 2E1, 2H9, 2D11, and 1F2 having ATCC Accession numbersPTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, andPTA-5959, respectively, and/or competes with the mouse monoclonalantibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, and 1F2 havingATCC Accession numbers, PTA-4591, PTA-4592, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively, as determined, e.g., inan ELISA assay or other appropriate competitive immunoassay, and alsobinds FcγRIIB with a greater affinity than said antibody or a fragmentthereof binds FcγRIIA.

The present invention also encompasses antibodies or fragments thereofcomprising an amino acid sequence of a variable heavy chain and/orvariable light chain that is at least 45%, at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% identical to theamino acid sequence of the variable heavy chain and/or light chain ofthe mouse monoclonal antibody produced by clone 8B5.3.4, 2B6, 3H7, 1D5,2E1, 2H9, 2D11, or 1F2 having ATCC accession numbers PTA-7610, PTA-4591,PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively. The present invention further encompasses antibodies orfragments thereof that specifically bind FcγRIIB with greater affinitythan said antibody or fragment thereof binds FcγRIIA, said antibodies orantibody fragments comprising an amino acid sequence of one or more CDRsthat is at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% identical to the amino acid sequenceof one or more CDRs of the mouse monoclonal antibody produced by clone8B5.3.4, 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 having ATCC accessionnumbers PTA-7610, PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively. The determination of percentidentity of two amino acid sequences can be determined by any methodknown to one skilled in the art, including BLAST protein searches.

The present invention also encompasses the use of antibodies or antibodyfragments that specifically bind FcγRIIB with greater affinity than saidantibodies or fragments thereof binds FcγRIIA, wherein said antibodiesor antibody fragments are encoded by a nucleotide sequence thathybridizes to the nucleotide sequence of the mouse monoclonal antibodyproduced by clone 8B5.3.4, 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 havingATCC accession numbers PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively, under stringent conditions. In apreferred embodiment, the invention provides antibodies or fragmentsthereof that specifically bind FcγRIIB with greater affinity than saidantibodies or fragments thereof bind FcγRIIA, said antibodies orantibody fragments comprising a variable light chain and/or variableheavy chain encoded by a nucleotide sequence that hybridizes understringent conditions to the nucleotide sequence of the variable lightchain and/or variable heavy chain of the mouse monoclonal antibodyproduced by clone 8B5.3.4, 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 havingATCC accession numbers PTA-7610, PTA-4591, PTA-4592, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively, under stringentconditions. In another preferred embodiment, the invention providesantibodies or fragments thereof that specifically bind FcγRIIB withgreater affinity than said antibodies or fragments thereof bind FcγRIIA,said antibodies or antibody fragments comprising one or more CDRsencoded by a nucleotide sequence that hybridizes under stringentconditions to the nucleotide sequence of one or more CDRs of the mousemonoclonal antibody produced by clone 8B5.3.4, 2B6, 3H7, 1D5, 2E1, 2H9,2D11, or 1F2 having ATCC accession numbers PTA-7610, PTA-4591, PTA-4592,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively.Stringent hybridization conditions include, but are not limited to,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., highly stringent conditions such as hybridization tofilter-bound DNA in 6×SSC at about 45° C. followed by one or more washesin 0.1×SSC/0.2% SDS at about 60° C., or any other stringenthybridization conditions known to those skilled in the art (see, forexample, Ausubel, F. M. et al., eds. 1989 Current Protocols in MolecularBiology, vol. 1, Green Publishing Associates, Inc. and John Wiley andSons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3, incorporated hereinby reference).

The constant domains of the antibodies may be selected with respect tothe proposed function of the antibody, in particular with regard to theeffector function which may be required. In some embodiments, theconstant domains of the antibodies are human IgA, IgE, IgG or IgMdomains.

The antibodies used in the methods of the invention include derivativesthat are modified, i.e, by the covalent attachment of any type ofmolecule to the antibody. For example, but not by way of limitation, theantibody derivatives include antibodies that have been modified, e.g.,by glycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

Further, the antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J.7:437-444; and Nissinoff, 1991, J. Immunol. 147:2429-2438). Theinvention provides methods employing the use of polynucleotidescomprising a nucleotide sequence encoding an antibody of the inventionor a fragment thereof.

The present invention encompasses single domain antibodies, includingcamelized single domain antibodies (See e.g., Muyldermans et al., 2001,Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech.1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25;International Publication Nos. WO 94/04678 and WO 94/25591; U.S. Pat.No. 6,005,079; which are incorporated herein by reference in theirentireties). In one embodiment, the present invention provides singledomain antibodies comprising two VH domains with modifications such thatsingle domain antibodies are formed.

The methods of the present invention also encompass the use ofantibodies or fragments thereof that have half-lives (e.g., serumhalf-lives) in a mammal, preferably a human, of greater than 15 days,preferably greater than 20 days, greater than 25 days, greater than 30days, greater than 35 days, greater than 40 days, greater than 45 days,greater than 2 months, greater than 3 months, greater than 4 months, orgreater than 5 months. The increased half-lives of the antibodies of thepresent invention or fragments thereof in a mammal, preferably a human,results in a higher serum titer of said antibodies or antibody fragmentsin the mammal, and thus, reduces the frequency of the administration ofsaid antibodies or antibody fragments and/or reduces the concentrationof said antibodies or antibody fragments to be administered. Antibodiesor fragments thereof having increased in vivo half-lives can begenerated by techniques known to those of skill in the art. For example,antibodies or fragments thereof with increased in vivo half-lives can begenerated by modifying (e.g., substituting, deleting or adding) aminoacid residues identified as involved in the interaction between the Fcdomain and the FcRn receptor. The antibodies of the invention may beengineered by methods described in Ward et al. to increase biologicalhalf-lives (See U.S. Pat. No. 6,277,375 B1). For example, antibodies ofthe invention may be engineered in the Fc-hinge domain to have increasedin vivo or serum half-lives.

Antibodies or fragments thereof with increased in vivo half-lives can begenerated by attaching to said antibodies or antibody fragments polymermolecules such as high molecular weight polyethyleneglycol (PEG). PEGcan be attached to said antibodies or antibody fragments with or withouta multifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of said antibodies or antibody fragments orvia epsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

The antibodies of the invention may also be modified by the methods andcoupling agents described by Davis et al. (See U.S. Pat. No. 4,179,337)in order to provide compositions that can be injected into the mammaliancirculatory system with substantially no immunogenic response.

The present invention also encompasses the use of antibodies or antibodyfragments comprising the amino acid sequence of any of the antibodies ofthe invention with mutations (e.g., one or more amino acidsubstitutions) in the framework or CDR regions. Preferably, mutations inthese antibodies maintain or enhance the avidity and/or affinity of theantibodies for FcγRIIIB to which they immunospecifically bind. Standardtechniques known to those skilled in the art (e.g., immunoassays) can beused to assay the affinity of an antibody for a particular antigen.

The invention further encompasses methods of modifying an effectorfunction of an antibody of the invention, wherein the method comprisesmodifying the carbohydrate content of the antibody using the methodsdisclosed herein or known in the art.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof. In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use human, chimeric orhumanized antibodies. Completely human antibodies are particularlydesirable for therapeutic treatment of human subjects. Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety.

5.1.1 Humanized Antibodies

In preferred embodiments, the antibodies are humanized antibodies. Ahumanized antibody is an antibody, a variant or a fragment thereof whichis capable of binding to a predetermined antigen and which comprises aframework region having substantially the amino acid sequence of a humanimmunoglobulin and a CDR having substantially the amino acid sequence ofa non-human immunoglobulin. A humanized FcγRIIB specific antibody maycomprise substantially all of at least one, and typically two, variabledomains in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody ofthe invention also comprises at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Theconstant domains of the humanized antibodies of the invention may beselected with respect to the proposed function of the antibody, inparticular the effector function which may be required. In someembodiments, the constant domains of the humanized antibodies of theinvention are human IgA, IgE, IgG or IgM domains. In a specificembodiment, human IgG constant domains, especially of the IgG1 and IgG3isotypes are used, when the humanized antibodies of the invention isintended for therapeutic uses and antibody effector functions areneeded. In alternative embodiments, IgG2 and IgG4 isotypes are used whenthe humanized antibody of the invention is intended for therapeuticpurposes and antibody effector function is not required. HumanizedFcγRIIB specific antibodies are disclosed in U.S. ProvisionalApplication Ser. Nos. 60/569,882 and 60/582,043, filed May 10, 2004 andJun. 21, 2004, respectively, and U.S. patent application Ser. No.11/126,978, filed May 10, 2005.

In some embodiments, the antibody contains both the light chain as wellas at least the variable domain of a heavy chain. In other embodiments,the antibody may further comprise one or more of the CH1, hinge, CH2,CH3, and CH4 regions of the heavy chain. The humanized antibody can beselected from any class of immunoglobulins, including IgM, IgG, IgD, IgAand IgE, and any isotype, including IgG₁, IgG₂, IgG₃ and IgG₄. In someembodiments, the constant domain is a complement fixing constant domainwhere it is desired that the humanized antibody exhibit cytotoxicactivity, and the class is typically IgG₁. In other embodiments, wheresuch cytotoxic activity is not desirable, the constant domain may be ofthe IgG₂ class. The humanized antibody may comprise sequences from morethan one class or isotype, and selecting particular constant domains tooptimize desired effector functions is within the ordinary skill in theart.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework may be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the donorantibody. Such mutations, however, are preferably not extensive.Usually, at least 75% of the humanized antibody residues will correspondto those of the parental framework region (FR) and CDR sequences, moreoften 90%, and most preferably greater than 95%. Humanized antibodiescan be produced using variety of techniques known in the art, includingbut not limited to, CDR-grafting (European Patent No. EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (European PatentNos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; and Roguska et al., 1994, Proc Natl Acad Sci USA91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniquesdisclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, 5,585,089,International Publication No. WO 9317105, Tan et al., 2002, J. Immunol.169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60, Morea et al.,2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem. 272:10678-84,Roguska et al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, CancerRes. 55 (23 Supp):5973s-5977s, Couto et al., 1995, Cancer Res.55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al., 1994, J.Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525, Riechmannet al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol.2:593-596. Often, framework residues in the framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; U.S.Publication Nos. 2004/0049014 and 2003/0229208; U.S. Pat. Nos.6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 andRiechmann et al., 1988, Nature 332:323, all of which are incorporatedherein by reference in their entireties.)

The present invention provides for the use of humanized antibodymolecules specific for FcγRIIB in which one or more regions of one ormore CDRs of the heavy and/or light chain variable regions of a humanantibody (the recipient antibody) have been substituted by analogousparts of one or more CDRs of a donor monoclonal antibody whichspecifically binds FcγRIIB, with a greater affinity than FcγRIIA, e.g.,a monoclonal antibody produced by clone 8B5.3.4, having ATCC accessionnumber PTA-7610, respectively. In other embodiments, the humanizedantibodies bind to the same epitope as the 8B5.3.4 antibody. In a mostpreferred embodiment, the humanized antibody specifically binds to thesame epitope as the donor murine antibody. It will be appreciated by oneskilled in the art that the invention encompasses CDR grafting ofantibodies in general. Thus, the donor and acceptor antibodies may bederived from animals of the same species and even same antibody class orsub-class. More usually, however, the donor and acceptor antibodies arederived from animals of different species. Typically the donor antibodyis a non-human antibody, such as a rodent MAb, and the acceptor antibodyis a human antibody.

In some embodiments, at least one CDR from the donor antibody is graftedonto the human antibody. In other embodiments, at least two andpreferably all three CDRs of each of the heavy and/or light chainvariable regions are grafted onto the human antibody. The CDRs maycomprise the Kabat CDRs, the structural loop CDRs or a combinationthereof. In some embodiments, the invention encompasses a humanizedFcγRIIB antibody comprising at least one CDR grafted heavy chain and atleast one CDR-grafted light chain.

In a preferred embodiment, the CDR regions of the humanized FcγRIIBspecific antibody are derived from a murine antibody specific forFcγRIIB In some embodiments, the humanized antibodies described hereincomprise alterations, including but not limited to amino acid deletions,insertions, modifications, of the acceptor antibody, i.e., human, heavyand/or light chain variable domain framework regions that are necessaryfor retaining binding specificity of the donor monoclonal antibody. Insome embodiments, the framework regions of the humanized antibodiesdescribed herein does not necessarily consist of the precise amino acidsequence of the framework region of a natural occurring human antibodyvariable region, but contains various alterations, including but notlimited to amino acid deletions, insertions, modifications that alterthe property of the humanized antibody, for example, improve the bindingproperties of a humanized antibody region that is specific for the sametarget as the murine FcγRIIB specific antibody. In most preferredembodiments, a minimal number of alterations are made to the frameworkregion in order to avoid large-scale introductions of non-humanframework residues and to ensure minimal immunogenicity of the humanizedantibody in humans. The donor monoclonal antibody is preferably amonoclonal antibody produced by clone 8B5.3.4 (having ATCC accessionnumber PTA-7610) which binds FcγRIIB

In a specific embodiment, the invention encompasses the use of aCDR-grafted antibody which specifically binds FcγRIIB with a greateraffinity than said antibody binds FcγRIIA, wherein the CDR-graftedantibody comprises a heavy chain variable region domain comprisingframework residues of the recipient antibody and residues from the donormonoclonal antibody, which specifically binds FcγRIIB with a greateraffinity than said antibody binds FcγRIIA, e.g., monoclonal antibodyproduced from clone 8B5.3.4. In another specific embodiment, theinvention encompasses the use of a CDR-grafted antibody whichspecifically binds FcγRIIB with a greater affinity than said antibodybinds FcγRIIA, wherein the CDR-grafted antibody comprises a light chainvariable region domain comprising framework residues of the recipientantibody and residues from the donor monoclonal antibody, whichspecifically binds FcγRIIB with a greater affinity than said antibodybinds FcγRIIA, e.g., monoclonal antibody produced from clones 8B5.3.4,2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2.

Preferably the humanized antibodies bind the extracellular domain ofnative human FcγRIIB The humanized anti-FcγRIIB antibodies of theinvention may have a heavy chain variable region comprising the aminoacid sequence of CDR1 (SEQ ID NO: 8) and/or CDR2 (SEQ ID NO: 9) and/orCDR3 (SEQ ID NO: 10) and/or a light chain variable region comprising theamino acid sequence of CDR1 (SEQ ID NO: 5) and/or a CDR2 (SEQ ID NO: 6)and/or CDR3 (SEQ ID NO: 7).

In a specific embodiment, the invention encompasses the use of ahumanized antibody comprising the CDRs of the 8B5.3.4 antibody in theprevention, treatment, management or amelioration of a B-cellmalignancy, or one or more symptoms thereof. In particular, an antibodywith the heavy chain variable domain having the amino acid sequence ofSEQ ID NO: 4 and the light chain variable domain having the amino acidsequence of SEQ ID NO: 3 is used in the prevention, treatment,management or amelioration of a B-cell malignancy, or one or moresymptoms thereof. In yet another preferred embodiment, the humanizedantibodies further do not bind Fc activation receptors, e.g., FcγIIIA,FcγIIIB, etc.

In one specific embodiment, a humanized 8B5.3.4 antibody is provided,wherein the VH region consists of the FR segments from the humangermline VH segment VH1-18 (Matsuda et al., 1998, J. Exp. Med.188:2151062) and JH6 (Ravetch et al., 1981, Cell 27(3 Pt. 2): 583-91),and one or more VH CDR regions of the 8B5.3.4 antibody, having the aminoacid sequence of SED ID NO: 8, SED ID NO: 9, or SED ID NO: 10. Inanother specific embodiment, the humanized 8B5.3.4 antibody furthercomprises a VL region, which consists of the FR segments of the humangermline VL segment VK-A26 (Lautner-Rieske et al., 1992, Eur. J.Immunol. 22:1023-1029) and JK4 (Hieter et al., 1982, J. Biol. Chem.257:1516-22), and one or more VL CDR regions of the 8B5.3.4 antibody,having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ IDNO: 7.

In particular, a humanized antibody is provided that immunospecificallybinds to extracellular domain of native human FcγRIIB, said antibodycomprising (or alternatively, consisting of) CDR sequences of the8B5.3.4 antibody, in any of the following combinations: a VH CDR1 and aVL CDR1; a VH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; a VH CDR2 anda VL CDR1; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and aVH CDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDR1, aVH CDR2 and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2; a VH CDR1, aVH CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1, a VH CDR2, aVH CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3; a VH CDR1, aVL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3; a VH CDR2, aVL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR3, aVL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, aVH CDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, a VH CDR3 and aVL CDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; a VH CDR1, a VHCDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VL CDR1 and a VLCDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VHCDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1 and a VLCDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VHCDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VLCDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VL CDR3; a VHCDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR2, a VHCDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combination thereof ofthe VH CDRs and VL CDRs disclosed herein.

5.1.2 Human Antibodies

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized using conventional methodologies with a selected antigen,e.g., all or a portion of a polypeptide of the invention. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93,which is incorporated herein by reference in its entirety). For adetailed discussion of this technology for producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g., International Publication Nos. WO 98/24893, WO96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126,5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598,which are incorporated by reference herein in their entirety. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.) and Medarex(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

5.1.3 Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. The present inventionprovides chimeric antibodies derived from antibodies produced fromhybridoma clones 8B5.3.4, 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 havingATCC accession numbers PTA-7610, PTA-4591, PTA-4592, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, 1985,Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al.,1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 6,311,415,5,807,715, 4,816,567, and 4,816,397, which are incorporated herein byreference in their entirety. Chimeric antibodies comprising one or moreCDRs from a non-human species and framework regions from a humanimmunoglobulin molecule can be produced using a variety of techniquesknown in the art including, for example, CDR-grafting (EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, PNAS91:969), and chain shuffling (U.S. Pat. No. 5,565,332). Each of theabove-identified references is incorporated herein by reference in itsentirety.

Often, framework residues in the framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323,which are incorporated herein by reference in their entireties.)

5.1.4 Fc Region Modifications

The invention encompasses antibodies with Fc constant domains comprisingone or more amino acid modifications which alter antibody effectorfunctions such as those disclosed in U.S. Patent Application PublicationNos. U.S. 2005/0037000 and U.S. 2005/0064514; U.S. Pat. Nos. 5,624,821and 5,648,260 and European Patent No. EP 0 307 434; all of which areincorporated herein by reference in their entireties. These antibodiesmay exhibit improved ADCC activity (i.e., 2-fold, 10-fold, 100-fold,500-fold, etc.) compared to comparable antibodies without amino acidmodification.

The present invention encompasses antibodies comprising modificationspreferably, in the Fc region that modify the binding affinity of theantibody to one or more FcγR. Methods for modifying antibodies withmodified binding to one or more FcγR are known in the art, see, e.g.,PCT Publication Nos. WO 04/029207, WO 04/029092, WO 04/028564, WO99/58572, WO 99/51642, WO 98/23289, WO 89/07142, WO 88/07089, and U.S.Pat. Nos. 5,843,597 and 5,642,821, each of which is incorporated hereinby reference in their entirety. In some embodiments, the inventionencompasses antibodies that have altered affinity for an activatingFcγR, e.g., FcγRIIIA Preferably such modifications also have an alteredFc-mediated effector function. Modifications that affect Fc-mediatedeffector function are known in the art (See U.S. Pat. No. 6,194,551,which is incorporated herein by reference in its entirety). The aminoacids that can be modified in accordance with the method of theinvention include but are not limited to Proline 329, Proline 331, andLysine 322. Proline 329, Proline 331 and Lysine 322 are preferablyreplaced with alanine, however, substitution with any other amino acidis contemplated. See International Publication No. WO 00/42072 and U.S.Pat. No. 6,194,551 which are incorporated herein by reference in theirentirety.

In one particular embodiment, the modification of the Fc regioncomprises one or more mutations in the Fc region. The one or moremutations in the Fc region may result in an antibody with an alteredantibody-mediated effector function, an altered binding to other Fcreceptors (e.g., Fc activation receptors), an altered ADCC activity, oran altered C1q binding activity, or an altered complement dependentcytotoxicity activity, or any combination thereof.

In some embodiments, the invention encompasses molecules comprising avariant Fc region having an amino acid modification at one or more ofthe following positions: 119, 125, 132, 133, 141, 142, 147, 149, 162,166, 185, 192, 202, 205, 210, 214, 215, 216, 217, 218, 219, 221, 222,223, 224, 225, 227, 229, 231, 232, 233, 235, 240, 241, 242, 243, 244,246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 258, 261, 262, 263,268, 269, 270, 272, 274, 275, 276, 279, 280, 281, 282, 284, 287, 288,289, 290, 291, 292, 293, 295, 298, 301, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 315, 316, 317, 318, 319, 320, 323, 326, 327,328, 330, 333, 334, 335, 337, 339, 340, 343, 344, 345, 347, 348, 352,353, 354, 355, 358, 359, 360, 361, 362, 365, 366, 367, 369, 370, 371,372, 375, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 404,406, 407, 408, 409, 410, 411, 412, 414, 415, 416, 417, 419, 420, 421,422, 423, 424, 427, 428, 431, 433, 435, 436, 438, 440, 441, 442, 443,446, or 447. Preferably, engineering of the Fc portion results inincreased cell-mediated killing and/or complement mediated killing ofthe tumor cells.

The invention encompasses molecules comprising variant Fc regionsconsisting of or comprising any of the mutations listed in the tablebelow in Table 2.

TABLE 2 EXEMPLARY MUTATIONS SINGLE SITE MUTANTS DOUBLE SITE MUTANTSK392R Q347H, A339V N315I S415I, L251F S132I K290E, L142P P396L G285E,P247H P396H K409R, S166N A162V E334A, K334A R292L R292L. K334E T359NK288N, A330S T366S R255L, E318K V379L F243L, E318K K288N V279L, P395SA330S K246T, Y319F F243L F243I, V379L E318K K288M, K334E V379M K334E,E308D S219Y E233D, K334E V282M K246T, P396H D401V H268D, E318D K222NK246I, K334N K334I K320E, K326E K334E S375C, P396L I377F K288N, K326NP247L P247L, N421K F372Y S298N, W381R K326E R255Q, K326E H224L V284A,F372L F275Y T394M. V397M L398V P247L, E389G K334N K290T, G371D S400PP247L, L398Q S407I P247L, I377F F372Y K326E, G385E T366N S298N, S407RK414N E258D, N384K M352L F241L, E258G T225S K370N, S440N I377N K317N,F423-DELETED K248M P227S, K290E R292G K334E, E380D S298N P291S, P353QD270E V240I, V281M E233G P232S, S304G P247L, L406F D399E, M428L L251F,F372L D399E, G402D D399E, M428L K392T, P396L H268N, P396L K326I, P396LH268D, P396L K210M, P396L L358P, P396L K334N, P396L V379M, P396L P227S,P396L P217S, P396L Q419H, P396L K370E, P396L L242F, P396L R255L, P396LV240A, P396L T250A, P396L P247S, P396L L410H, P396L Q419L, P396L V427A,P396L E258D, P396L N384K, P396L V323I, P396L P244H, P396L V305L, P396LS400F, P396L V303I, P396L A330V, Q419H V263Q, E272D K326E, A330T

In yet other embodiments, the invention encompasses molecules comprisingvariant Fc regions having more than two amino acid modifications. Anon-limiting example of such variants is listed in the table below(Table 3). The invention encompasses mutations listed in Table 3 whichfurther comprise one or more amino acid modifications such as thosedisclosed herein.

TABLE 3 EXEMPLARY COMBINATION VARIANTS D399E, R292L, V185M R301C, M252L,S192T P291S, K288E, H268L, A141V S383N, N384K, T256N, V262L, K218E,R214I, K205E, F149Y, K133M S408I, V215I, V125L G385E, P247H V348M,K334N, F275I, Y202M, K147T H310Y, T289A, Y407V, E258D R292L, P396L,T359N F275I, K334N, V348M F243L. R255L, E318K K334E, T359N, T366S T256S,V305I, K334E, N390S T335N, K370E, A378V, T394M, S424L K334E, T359N,T366S, Q386R K288N, A330S, P396L P244H, L358M, V379M, N384K, V397MP217S, A378V, S408R P247L, I253N, K334N D312E, K327N, I378S D280E,S354F, A431D, L441I K218R, G281D, G385R P247L, A330T, S440G T355N,P387S, H435Q P247L, A431V, S442F P343S, P353L, S375I, S383N E216D,E345K, S375I K288N, A330S, P396L K222N, T335N, K370E, A378V, T394MG316D, A378V, D399E N315I, V379M, T394M K326Q, K334E, T359N, T366SA378V, N390I, V422I V282E, V369I, L406F V397M, T411A, S415N T223I,T256S, L406F L235P, V382M, S304G, V305I, V323I P247L, W313R, E388GD221Y, M252I, A330G, A339T, T359N, V422I, H433L F243I, V379L, G420VA231V, Q386H, V412M T215P, K274N, A287G, K334N, L365V, P396L P244A,K326I, C367R, S375I, K447T R301H, K340E, D399E C229Y, A287T, V379M,P396L, L443V E269K, K290N, Q311R, H433Y E216D, K334R, S375I T335N,P387S, H435Q K246I, Q362H, K370E K334E, E380D, G446V V303I, V369F, M428LK246E, V284M, V308A E293V, Q295E, A327T Y319F, P352L, P396L D221E,D270E, V308A, Q311H, P396L, G402D K290T, N390I, P396L K288R, T307A,K344E, P396L V273I, K326E, L328I, P396L K326I, S408N, P396L K261N,K210M, P396L F243L, V305I, A378D, F404S, P396L K290E, V369A, T393A,P396L K210N, K222I, K320M, P396L P217S, V305I, I309L, N390H, P396LK246N, Q419R, P396L P217A, T359A, P396L V215I, K290V, P396L F275L,Q362H, N384K, P396L A330V, H433Q, V427M V263Q, E272D, Q419H N276Y,T393N, W417R V282L, A330V, H433Y, T436R V284M, S298N, K334E, R355WA330V, G427M, K438R S219T, T225K, D270E, K360R K222E, V263Q, S298NE233G, P247S, L306P S219T, T225K, D270E S254T, A330V, N361D, P243LV284M, S298N, K334E, R355W R416T D270E, G316D, R416G K392T, P396L, D270ER255L, P396L, D270E V240A, P396L, D270E Q419H, P396L, D270E K370E,P396L, D270E P247L, N421K, D270E R292P, V305I R292P, V305I, F243L V284M,R292L, K370N F243L, R292L, Y300L

In most preferred embodiments, an anti-FcγRIIB antibody of the inventionhas a modified Fc region with altered affinity for activating and/orinhibitory receptors, wherein the Fc domain has one or more amino acidmodifications, wherein said one or more amino acid modifications is asubstitution at position 288 with asparagine, at position 330 withserine and at position 396 with leucin (MgFc10) (see, Tables 2 and 3);or a substitution at position 334 with glutamic acid, at position 359with asparagine, and at position 366 with serine (MgFc13); or asubstitution at position 316 with aspartic acid, at position 378 withvaline, and at position 399 with glutamic acid (MgFc27); or asubstitution at position 392 with threonine, and at position 396 withleucine (MgFc38); or a substitution at position 221 with glutamic acid,at position 270 with glutamic acid, at position 308 with alanine, atposition 311 with histidine, at position 396 with leucine, and atposition 402 with aspartic acid (MgFc42); or a substitution at position240 with alanine, and at position 396 with leucine (MgFc52); or asubstitution at position 410 with histidine, and at position 396 withleucine (MgFc53); or a substitution at position 243 with leucine, atposition 305 with isoleucine, at position 378 with aspartic acid, atposition 404 with serine, and at position 396 with leucine (MgFc54); ora substitution at position 255 with leucine, and at position 396 withleucine (MgFc55); or a substitution at position 370 with glutamic acidand at position 396 with leucine (MgFc59); or a substitution at position243 with leucine, at position 292 with proline, at position 300 withleucine, at position 305 with isoleucine, and at position 396 withleucine (MgFc88); or a substitution at position 243 with leucine, atposition 292 with proline, at position 300 with leucine, and at position396 with leucine (MgFc88A); or a substitution at position 243 withleucine, at position 292 with proline, and at position 300 with leucine(MgFc155). (See, also, Tables 2, 3A and 3B of U.S. Patent ApplicationPublication No. 2005/0064514 A1, to Stavenhagen et al., filed Jul. 28,2004, which is herein incorporated by reference.)

In a preferred embodiment, the anti-FcγRIIB antibody, such as the8B5.3.4 antibody, has a modified Fc region with a leucine at position243, a proline at position 292, a leucine at position 300, an isoleucineat position 305 and a leucine at position 396.

In specific embodiments, the variant Fc region has a leucine at position247, a lysine at position 421 and a glutamic acid at position 270(MgFc31/60); a threonine at position 392, a leucine at position 396, anda glutamic acid at position 270 (MgFc38/60); a threonine at position392, a leucine at position 396, a glutamic acid at position 270, and aleucine at position 243 (MgFc38/60/F243L); a histidine at position 419,a leucine at position 396, and a glutamic acid at position 270(MGFc51/60); a histidine at position 419, a leucine at position 396, aglutamic acid at position 270, and a leucine at position 243(MGFc51/60/F243L); a lysine at position 255, a leucine at position 396,and a glutamic acid at position 270 (MGFc55/60); a lysine at position255, a leucine at position 396, a glutamic acid at position 270, and alysine at position 300 (MGFc55/60/Y300L); a lysine at position 255, aleucine at position 396, a glutamic acid at position 270, and a leucineat position 243 (MgFc55/60/F243L); a glutamic acid at position 370, aleucine at position 396, and a glutamic acid at position 270(MGFc59/60); a glutamic acid at position 270, an aspartic acid atposition 316, and a glycine at position 416 (MgFc71); a leucine atposition 243, a proline at position 292, an isoleucine at position 305,and a leucine at position 396 (MGFc74/P396L); a glutamine at position297, or any combination of the individual substitutions.

5.1.5 Carbohydrate Modifications

The invention also provides antibodies with altered oligosaccharidecontent. Oligosaccharides as used herein refer to carbohydratescontaining two or more simple sugars and the two terms may be usedinterchangeably herein. Carbohydrate moieties of the instant inventionwill be described with reference to commonly used nomenclature in theart. For a review of carbohydrate chemistry, see, e.g., Hubbard et al.,1981 Ann. Rev. Biochem., 50: 555-583, which is incorporated herein byreference in its entirety. This nomenclature includes for example, Manwhich represents mannose; GlcNAc which represents 2-N-acetylglucosamine;Gal which represents galactose; Fuc for fucose and Glc for glucose.Sialic acids are described by the shorthand notation NeuNAc for5-N-acetylneuraminic acid, and NeuNGc for 5-glycolneuraminic.

In general, antibodies contain carbohydrate moeities at conservedpositions in the constant region of the heavy chain, and up to 30% ofhuman IgGs have a glycosylated Fab region. IgG has a single N-linkedbiantennary carbohydrate structure at Asn 297 which resides in the CH2domain (Jefferis et al., 1998, Immunol. Rev. 163: 59-76; Wright et al.,1997, Trends Biotech 15: 26-32). Human IgG typically has a carbohydrateof the following structure; GlcNAc(Fucose)-GlcNAc-Man-(ManGlcNAc)₂.However variations among IgGs in carbohydrate content does occur whichleads to altered function, see, e.g., Jassal et al., 2001 Biochem.Biophys. Res. Commun. 288: 243-9; Groenink et al., 1996 J. Immunol. 26:1404-7; Boyd et al., 1995 Mol. Immunol. 32: 1311-8; Kumpel et al., 1994,Human Antibody Hybridomas, 5: 143-51. The invention encompassesantibodies comprising a variation in the carbohydrate moiety that isattached to Asn 297. In one embodiment, the carbohydrate moiety has agalactose and/or galactose-sialic acid at one or both of the terminalGlcNAc and/or a third GlcNac arm (bisecting GlcNAc).

In some embodiments, the antibodies of the invention are substantiallyfree of one or more selected sugar groups, e.g., one or more sialic acidresidues, one or more galactose residues, one or more fucose residues.An antibody that is substantially free of one or more selected sugargroups may be prepared using common methods known to one skilled in theart, including for example recombinantly producing an antibody of theinvention in a host cell that is defective in the addition of theselected sugar groups(s) to the carbohydrate moiety of the antibody,such that about 90-100% of the antibody in the composition lacks theselected sugar group(s) attached to the carbohydrate moiety. Alternativemethods for preparing such antibodies include for example, culturingcells under conditions which prevent or reduce the addition of one ormore selected sugar groups, or post-translational removal of one or moreselected sugar groups.

In a specific embodiment, the invention encompasses a method ofproducing a substantially homogenous antibody preparation, wherein about80-100% of the antibody in the composition lacks a fucose on itscarbohydrate moiety, e.g., the carbohydrate attachment on Asn 297. Theantibody may be prepared for example by (a) use of an engineered hostcell that is deficient in fucose metabolism such that it has a reducedability to fucosylate proteins expressed therein; (b) culturing cellsunder conditions which prevent or reduce fusocylation; (c)post-translational removal of fucose, e.g., with a fucosidase enzyme; or(d) purification of the antibody so as to select for the product whichis not fucosylated. Most preferably, nucleic acid encoding the desiredantibody is expressed in a host cell that has a reduced ability tofucosylate the antibody expressed therein. Preferably the host cell is adihydrofolate reductase deficient chinese hamster ovary cell (CHO),e.g., a Lec 13 CHO cell (lectin resistant CHO mutant cell line; Ribka &Stanley, 1986, Somatic Cell & Molec. Gen. 12(1): 51-62; Ripka et al.,1986 Arch. Biochem. Biophys. 249(2): 533-45), CHO-K1, DUX-B11, CHO-DP12or CHO-DG44, which has been modified so that the antibody is notsubstantially fucosylated. Thus, the cell may display altered expressionand/or activity for the fucoysltransferase enzyme, or another enzyme orsubstrate involved in adding fucose to the N-linked oligosaccharide sothat the enzyme has a diminished activity and/or reduced expressionlevel in the cell. For methods to produce antibodies with altered fucosecontent, see, e.g., WO 03/035835 and Shields et al., 2002, J. Biol.Chem. 277(30): 26733-40; both of which are incorporated herein byreference in their entirety.

In some embodiments, the altered carbohydrate modifications modulate oneor more of the following: solubilization of the antibody, facilitationof subcellular transport and secretion of the antibody, promotion ofantibody assembly, conformational integrity, and antibody-mediatedeffector function. In a specific embodiment the altered carbohydratemodifications enhance antibody mediated effector function relative tothe antibody lacking the carbohydrate modification. Carbohydratemodifications that lead to altered antibody mediated effector functionare well known in the art (for e.g., see Shields R. L. et al., 2001, J.Biol. Chem. 277(30): 26733-40; Davies J. et al., 2001, Biotechnology &Bioengineering, 74(4): 288-294). In another specific embodiment, thealtered carbohydrate modifications enhance the binding of antibodies ofthe invention to FcγRIIB receptor. Altering carbohydrate modificationsin accordance with the methods of the invention includes, for example,increasing the carbohydrate content of the antibody or decreasing thecarbohydrate content of the antibody. Methods of altering carbohydratecontents are known to those skilled in the art, see, e.g., Wallick etal., 1988, Journal of Exp. Med. 168(3): 1099-1109; Tao et al., 1989Journal of Immunology, 143(8): 2595-2601; Routledge et al., 1995Transplantation, 60(8): 847-53; Elliott et al. 2003; NatureBiotechnology, 21: 414-21; Shields et al. 2002 Journal of BiologicalChemistry, 277(30): 26733-40; all of which are incorporated herein byreference in their entirety.

In some embodiments, the invention encompasses antibodies comprising oneor more glycosylation sites, so that one or more carbohydrate moietiesare covalently attached to the antibody. In other embodiments, theinvention encompasses antibodies comprising one or more glycosylationsites and one or more modifications in the Fc region, such as thosedisclosed supra and those known to one skilled in the art. In preferredembodiments, the one or more modifications in the Fc region enhance theaffinity of the antibody for an activating FcγR, e.g., FcγRIIIA,relative to the antibody comprising the wild type Fc regions. Antibodiesof the invention with one or more glycosylation sites and/or one or moremodifications in the Fc region have an enhanced antibody mediatedeffector function, e.g., enhanced ADCC activity. In some embodiments,the invention further comprises antibodies comprising one or moremodifications of amino acids that are directly or indirectly known tointeract with a carbohydrate moiety of the antibody, including but notlimited to amino acids at positions 241, 243, 244, 245, 245, 249, 256,258, 260, 262, 264, 265, 296, 299, and 301. Amino acids that directly orindirectly interact with a carbohydrate moiety of an antibody are knownin the art, see, e.g., Jefferis et al., 1995 Immunology Letters, 44:111-7, which is incorporated herein by reference in its entirety.

The invention encompasses antibodies that have been modified byintroducing one or more glycosylation sites into one or more sites ofthe antibodies, preferably without altering the functionality of theantibody, e.g., binding activity to FcγRIIB Glycosylation sites may beintroduced into the variable and/or constant region of the antibodies ofthe invention. As used herein, “glycosylation sites” include anyspecific amino acid sequence in an antibody to which an oligosaccharide(i.e., carbohydrates containing two or more simple sugars linkedtogether) will specifically and covalently attach. Oligosaccharide sidechains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of anoligosaccharide moiety to the side chain of an asparagine residue.O-linked glycosylation refers to the attachment of an oligosaccharidemoiety to a hydroxyamino acid, e.g., serine, threonine. The antibodiesof the invention may comprise one or more glycosylation sites, includingN-linked and O-linked glycosylation sites. Any glycosylation site forN-linked or O-linked glycosylation known in the art may be used inaccordance with the instant invention. An exemplary N-linkedglycosylation site that is useful in accordance with the methods of thepresent invention, is the amino acid sequence: Asn-X-Thr/Ser, wherein Xmay be any amino acid and Thr/Ser indicates a threonine or a serine.Such a site or sites may be introduced into an antibody of the inventionusing methods well known in the art to which this invention pertains.See, for example, “In vitro Mutagenesis,” Recombinant DNA: A ShortCourse, J. D. Watson, et al. W. H. Freeman and Company, New York, 1983,chapter 8, pp. 106-116, which is incorporated herein by reference in itsentirety. An exemplary method for introducing a glycosylation site intoan antibody of the invention may comprise: modifying or mutating anamino acid sequence of the antibody so that the desired Asn-X-Thr/Sersequence is obtained.

In some embodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by adding ordeleting a glycosylation site. Methods for modifying the carbohydratecontent of antibodies are well known in the art and encompassed withinthe invention, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S.Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No.2003/0115614; U.S. Pat. Nos. 6,218,149; 6,472,511; all of which areincorporated herein by reference in their entirety. In otherembodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by deleting one ormore endogenous carbohydrate moieties of the antibody.

In some specific embodiments, the invention encompasses the use ofmodified FcγRIIB antibodies wherein the N-glycosylation consensus siteAsn₅₀-Val-Ser of the CDR2 region has been modified, so that theglycosylation site at position 50 is eliminated. Although not intendingto be bound by a particular mechanism of action, removal of theglycosylation site may limit potential variation in production of theantibody as well as potential immunogenicity in a pharmaceuticalapplication. In a specific embodiment, the invention encompasses the useof a humanized FcγRIIB antibody wherein the amino acid at position 50has been modified, e.g., deleted or substituted. In another specificembodiment, the invention further encompasses the use of an antibodywith an amino acid modification, e.g., deletion or substitution, atposition 51. In one specific embodiment, the invention encompasses theuse of a humanized FcγRIIB antibody wherein the amino acid at position50 has been replaced with tyrosine. In another more specific embodiment,the invention encompasses the use of a FcγRIIB antibody wherein theamino acid at position 50 has been replaced with tyrosine and the aminoacid at position 51 has been replaced with alanine.

5.1.6 FcγRIIB Agonists and Antagonists

In addition to the use of a FcγRIIB-specific antibody, an analog,derivative, or an antigen-binding fragment thereof in the methods andcompositions of the invention, other FcγRIIB agonist and antagonists maybe used in accordance with the methods of the invention. FcγRIIBagonists and antagonists include, but are not limited to, proteinaceousmolecules (e.g., proteins, polypeptides (e.g., soluble FcγRIIBpolypeptides), peptides, fusion proteins (e.g., soluble FcγRIIBpolypeptides conjugated to a therapeutic moiety), nucleic acid molecules(e.g., FcγRIIB antisense nucleic acid molecules, triple helices, dsRNAthat mediates RNAi, or nucleic acid molecules encoding proteinaceousmolecules), organic molecules, inorganic molecules, small organicmolecules, drugs, and small inorganic molecules that block, inhibit,reduce or neutralize a function, an activity and/or the expression of aFcγRIIB polypeptide, expressed by an immune cell, preferably a B-cell.In some embodiments, a FcγRIIB agonist or antagonist used in accordancewith the methods of the invention is not a small organic molecule, adrug or an antisense molecule. FcγRIIB agonists and antagonists can beidentified using techniques well-known in the art or described herein.

Prophylactic and therapeutic compounds of the invention include, but arenot limited to, proteinaceous molecules, including, but not limited to,peptides, polypeptides, proteins, including post-translationallymodified proteins, antibodies, etc.; small molecules (less than 1000daltons), inorganic or organic compounds; nucleic acid moleculesincluding, but not limited to, double-stranded or single-stranded DNA,double-stranded or single-stranded RNA, as well as triple helix nucleicacid molecules. Prophylactic and therapeutic compounds can be derivedfrom any known organism (including, but not limited to, animals, plants,bacteria, fungi, and protista, or viruses) or from a library ofsynthetic molecules.

In certain embodiments, FcγRIIB antagonists reduce a function, activity,and/or expression of a FcγRIIB polypeptide in a subject with a B-cellmalignancy. In other embodiments, the FcγRIIB antagonists directly bindto a FcγRIIB polypeptide and directly or indirectly modulate an activityand/or function of B-lymphocytes. In particular embodiments, FcγRIIBantagonists inhibit or reduce B-cell proliferation in a subject with aB-cell malignancy as determined by standard in vivo and/or in vitroassays described herein or well-known to those skilled in the art. In aspecific embodiment, FcγRIIB antagonists mediate the depletion oflymphocytes, in particular peripheral blood B-cells, in a subject with aB-cell malignancy as determined by standard in vivo and/or in vitroassays described herein or well-known to those skilled in the art. Inanother embodiment, FcγRIIB antagonists directly or indirectly modulatean activity and/or function of B-lymphocytes by utilizingantibody-dependent cell-mediated cytotoxicity (ADCC).

In a preferred embodiment, proteins, polypeptides or peptides (includingantibodies and fusion proteins) that are utilized as FcγRIIB antagonistsare derived from the same species as the recipient of the proteins,polypeptides or peptides so as to reduce the likelihood of an immuneresponse to those proteins, polypeptides or peptides. In anotherpreferred embodiment, when the subject is a human, the proteins,polypeptides, or peptides that are utilized as FcγRIIB antagonists arehuman or humanized.

Nucleic acid molecules encoding proteins, polypeptides, or peptides thatfunction as FcγRIIB antagonists can be administered to a subject with aB-cell malignancy, in accordance with the methods of the invention.Further, nucleic acid molecules encoding derivatives, analogs, fragmentsor variants of proteins, polypeptides, or peptides that function asFcγRIIB antagonists can be administered to a subject with a B-cellmalignancy in accordance with the methods of the invention. Preferably,such derivatives, analogs, variants and fragments retain the FcγRIIBantagonist activity of the full-length wild-type protein, polypeptide,or peptide.

5.2 Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide; or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide) togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. Antibodies may be usedfor example to target heterologous polypeptides to particular celltypes, either in vitro or in vivo, by fusing or conjugating theantibodies to antibodies specific for particular cell surface receptors.Antibodies fused or conjugated to heterologous polypeptides may also beused in in vitro immunoassays and purification methods using methodsknown in the art. See e.g., PCT Publication No. WO 93/21232; EP 439,095;Naramura et al., 1994, Immunol. Lett., 39:91-99; U.S. Pat. No.5,474,981; Gillies et al., 1992, Proc Natl Acad Sci, 89:1428-1432; andFell et al., 1991, J. Immunol., 146:2446-2452, each of which isincorporated herein by reference in their entireties.

Further, an antibody may be conjugated to a therapeutic agent or drugmoiety that modifies a given biological response. Therapeutic agents ordrug moieties are not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, a toxin such as abrin, ricin A, pseudomonasexotoxin (i.e., PE-40), or diphtheria toxin, ricin, gelonin, andpokeweed antiviral protein, a protein such as tumor necrosis factor,interferons including, but not limited to, α-interferon (IFN-α),β-interferon (IFN-β), nerve growth factor (NGF), platelet derived growthfactor (PDGF), tissue plasminogen activator (TPA), an apoptotic agent(e.g., TNF-α, TNF-β, AIM I as disclosed in PCT Publication No. WO97/33899), AIM II (see, e.g., PCT Publication No. WO 97/34911), FasLigand (Takahashi et al., 1994 J. Immunol., 6:1567-1574), and VEGI (PCTPublication No. WO 99/23105), a thrombotic agent or an anti-angiogenicagent (e.g., angiostatin or endostatin), or a biological responsemodifier such as, for example, a lymphokine (e.g., interleukin-1(“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocytemacrophage colony stimulating factor (“GM-CSF”), and granulocyte colonystimulating factor (“G-CSF”)), macrophage colony stimulating factor,(“M-CSF”), or a growth factor (e.g., growth hormone (“GH”); a protease,or a ribonuclease).

Antibodies can be fused to marker sequences, such as a peptide, tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., 1989 Proc. Natl. Acad. Sci. USA, 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., 1984Cell, 37:767) and the “flag” tag (Knappik et al., 1994 Biotechniques,17(4):754-761).

The present invention further includes the use of compositionscomprising heterologous polypeptides fused or conjugated to antibodyfragments. For example, the heterologous polypeptides may be fused orconjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment,or portion thereof. Methods for fusing or conjugating polypeptides toantibody portions are known in the art. See, e.g., U.S. Pat. Nos.5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP307,434; EP 367,166; International Publication Nos. WO 96/04388 and WO91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil etal., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341 (said referencesincorporated by reference in their entireties).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16:76; Hansson, et al., 1999, J. Mol. Biol. 287:265; andLorenzo and Blasco, 1998, BioTechniques 24:308 (each of these patentsand publications are hereby incorporated by reference in its entirety).Antibodies or fragments thereof, or the encoded antibodies or fragmentsthereof, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. One or more portions of a polynucleotide encoding anantibody or antibody fragment, which portions specifically bind toFcγRIIB may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

The present invention also encompasses antibodies conjugated to adiagnostic or therapeutic agent or any other molecule for which serumhalf-life is desired to be increased. The antibodies can be useddiagnostically to, for example, monitor the development or progressionof a disease, disorder or infection as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals, and nonradioactive paramagnetic metal ions. The detectablesubstance may be coupled or conjugated either directly to the antibodyor indirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as diagnostics according to the present invention.Such diagnosis and detection can be accomplished by coupling theantibody to detectable substances including, but not limited to, variousenzymes, enzymes including, but not limited to, horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;prosthetic group complexes such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent material such as, but not limitedto, luminol; bioluminescent materials such as, but not limited to,luciferase, luciferin, and aequorin; radioactive material such as, butnot limited to, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt(⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu),manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorous(³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re),rhodium (¹⁰⁵Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc),selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc),thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions.

An antibody may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Cytotoxins or cytotoxic agents include any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive materials or macrocyclic chelators useful for conjugatingradiometal ions (see above for examples of radioactive materials). Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50each incorporated by reference in their entireties.

Techniques for conjugating such therapeutic moieties to antibodies arewell known; see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R.Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp.623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp.475-506); “Analysis, Results, And Future Prospective Of The TherapeuticUse Of Radiolabeled Antibody In Cancer Therapy”, in MonoclonalAntibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),1985, pp. 303-16, Academic Press; and Thorpe et al., Immunol. Rev.,62:119-58, 1982.

An antibody or fragment thereof, with or without a therapeutic moietyconjugated to it, administered alone or in combination with cytotoxicfactor(s) and/or cytokine(s) can be used as a therapeutic.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

5.3 Preparation and Characterization of Monoclonal Antibodies of theInvention

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

In one particular embodiment, the invention provides a method forproducing monoclonal antibodies that specifically bind FcγRIIB withgreater affinity than said monoclonal antibodies bind FcγRIIAcomprising: immunizing one or more FcγRIIA transgenic mice (See U.S.Pat. Nos. 5,877,396 and 5,824,487) with the purified extracellulardomain of human FcγRIIB, amino acids 1-180; producing hybridoma celllines from spleen cells of said mice, screening said hybridoma cellslines for one or more hybridoma cell lines that produce antibodies thatspecifically bind FcγRIIB with greater affinity than said antibodiesbind FcγRIIA. In another specific embodiment, the invention provides amethod for producing FcγRIIB monoclonal antibodies that specificallybind FcγRIIB, particularly human FcγRIIB, with a greater affinity thansaid monoclonal antibodies bind FcγRIIA, said method further comprising:immunizing one or more FcγRIIA transgenic mice with purified FcγRIIB oran immunogenic fragment thereof, booster immunizing said mice sufficientnumber of times to elicit an immune response, producing hybridoma cellslines from spleen cells of said one or more mice, screening saidhybridoma cell lines for one or more hybridoma cell lines that produceantibodies that specifically bind FcγRIIB with a greater affinity thansaid antibodies bind FcγRIIA. In one embodiment of the invention, saidmice are immunized with purified FcγRIIB which has been mixed with anyadjuvant known in the art to enhance immune response. Adjuvants that canbe used in the methods of the invention include, but are not limited to,protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG,Corynebacterium parvum, Salmonella minnesota) and bacterial componentsincluding cell wall skeleton, trehalose dimycolate, monophosphoryl lipidA, methanol extractable residue (MER) of tubercle bacillus, complete orincomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g.,aluminum hydroxide, iodoacetate and cholesteryl hemisuccinateor; nakedDNA adjuvants. Other adjuvants that can be used in the methods of theinvention include, Cholera toxin, paropox proteins, MF-59 (ChironCorporation; See also Bieg et al., 1999, Autoimmunity, 31(1):15-24,which is incorporated herein by reference), MPL® (Corixa Corporation;See also Lodmell D. I. et al., 2000 Vaccine, 18: 1059-1066; Ulrich etal., 2000, Methods in Molecular Medicine, 273-282; Johnson et al., 1999,Journal of Medicinal Chemistry, 42: 4640-4649; Baldridge et al., 1999Methods, 19: 103-107, all of which are incorporated herein byreference), RC-529 adjuvant (Corixa Corporation; the lead compound fromCorixa's aminoalkyl glucosaminide 4-phosphate (AGP) chemical library,see also www.corixa.com), and DETOX™ adjuvant (Corixa Corporation;DETOX™ adjuvant includes MPL® adjuvant (monophosphoryl lipid A) andmycobacterial cell wall skeleton; See also Eton et al., 1998, Clin.Cancer Res, 4(3):619-27; and Gubta R. et al., 1995, Vaccine,13(14):1263-76 both of which are incorporated herein by reference.)

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CH1 region and at least a portion of the hingeregion of the heavy chain.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In a particularembodiment, such phage can be utilized to display antigen bindingdomains, such as Fab and Fv or disulfide-bond stabilized Fv, expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phage,including fd and M13. The antigen binding domains are expressed as arecombinantly fused protein to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theimmunoglobulins, or fragments thereof, of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods, 182:41-50,1995; Ames et al., J. Immunol. Methods, 184:177-186, 1995; Kettleboroughet al., Eur. J. Immunol., 24:952-958, 1994; Persic et al., Gene,187:9-18, 1997; Burton et al., Advances in Immunology, 57:191-280, 1994;PCT Application No. PCT/GB91/01134; PCT Publications WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which isincorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT Publication WO92/22324; Mullinax et al., BioTechniques, 12(6):864-869, 1992; and Sawaiet al., AJRI, 34:26-34, 1995; and Better et al., Science, 240:1041-1043,1988 (each of which is incorporated by reference in its entirety).Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu etal., Proc Natl Acad Sci USA, 90:7995-7999, 1993; and Skerra et al.,Science, 240:1038-1040, 1988.

Phage display technology can be used to increase the affinity of anantibody of the invention for FcγRIIB This technique would be useful inobtaining high affinity antibodies that could be used in thecombinatorial methods of the invention. The technology, referred to asaffinity maturation, employs mutagenesis or CDR walking and re-selectionusing FcγRIIB or an antigenic fragment thereof to identify antibodiesthat bind with higher affinity to the antigen when compared with theinitial or parental antibody (See, e.g., Glaser et al., 1992, J.Immunology 149:3903). Mutagenizing entire codons rather than singlenucleotides results in a semi-randomized repertoire of amino acidmutations. Libraries can be constructed consisting of a pool of variantclones each of which differs by a single amino acid alteration in asingle CDR and which contain variants representing each possible aminoacid substitution for each CDR residue. Mutants with increased bindingaffinity for the antigen can be screened by contacting the immobilizedmutants with labeled antigen. Any screening method known in the art canbe used to identify mutant antibodies with increased avidity to theantigen (e.g., ELISA) (See Wu et al., 1998, Proc Natl. Acad Sci. USA95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDR walking whichrandomizes the light chain is also possible (See Schier et al., 1996, J.Mol. Bio. 263:551).

Antibodies of the invention may be further characterized by epitopemapping, so that antibodies may be selected that have the greatestspecificity for FcγRIIB compared to FcγRIIA. Epitope mapping methods ofantibodies are well known in the art and encompassed within the methodsof the invention. In certain embodiments fusion proteins comprising oneor more regions of FcγRIIB may be used in mapping the epitope of anantibody of the invention. In a specific embodiment, the fusion proteincontains the amino acid sequence of a region of an FcγRIIB fused to theFc portion of human IgG2. Each fusion protein may further comprise aminoacid substitutions and/or replacements of certain regions of thereceptor with the corresponding region from a homolog receptor, e.g.,FcγRIIA, as shown in Table 4 below. pMGX125 and pMGX132 contain the IgGbinding site of the FcγRIIB receptor, the former with the C-terminus ofFcγRIIB and the latter with the C-terminus of FcγRIIA and can be used todifferentiate C-terminus binding. The others have FcγRIIA substitutionsin the IgG binding site and either the FcγIIA or FcγIIB N-terminus.These molecules can help determine the part of the receptor moleculewhere the antibodies bind.

TABLE 4List of the fusion proteins that may be used to investigate the epitopeof the monoclonal anti-FcγRIIB antibodies. Residues 172 to 180 belong to theIgG binding site of FcγRIIA and B. The specific amino acids from FcγRIIAsequence are in bold. The C-terminus sequence APSSS is SEQ ID NO: 11 and theC-terminus sequence VPSMGSSS is SEQ ID NO: 12. N- SEQ ID C- SEQ IDPlasmid Receptor terminus 172-180 NO: terminus NO: pMGX125 RIIb IIbKKFSRSDPN 13 APS------SS(IIb) 11 pMGX126 RIIa/b IIa QKFSRLDPN 14APS------SS(IIb) 11 pMGX127 IIa QKFSRLDPT 15 APS------SS(IIb) 11 pMGX128IIb KKFSRLDPT 16 APS------SS(IIb) 11 pMGX129 IIa QKFSHLDPT 17APS------SS(IIb) 11 pMGX130 IIb KKFSHLDPT 18 APS------SS(IIb) 11 pMGX131IIa QKFSRLDPN 19 VPSMGSSS(IIa) 12 pMGX132 IIb KKFSRSDPN 13 VPSMGSSS(IIa)12 pMGX133 RIIa-131R IIa QKFSRLDPT 15 VPSMGSSS(IIa) 12 pMGX134 RIIa-131HIIa QKFSHLDPT 17 VPSMGSSS(IIa) 12 pMGX135 IIb KKFSRLDPT 16 VPSMGSSS(IIa)12 pMGX136 IIb KKFSHLDPT 18 VPSMGSSS(IIa) 12

The fusion proteins may be used in any biochemical assay fordetermination of binding to an anti-FcγRIIB antibody of the invention,e.g., an ELISA. In other embodiments, further confirmation of theepitope specificity may be done by using peptides with specific residuesreplaced with those from the FcγRIIA sequence.

The antibodies of the invention may be characterized for specificbinding to FcγRIIB using any immunological or biochemical based methodknown in the art for characterizing including quantitating, theinteraction of the antibody to FcγRIIB Specific binding of an antibodyof the invention to FcγRIIB may be determined for example usingimmunological or biochemical based methods including, but not limitedto, an ELISA assay, surface plasmon resonance assays,immunoprecipitation assay, affinity chromatography, and equilibriumdialysis. Immunoassays which can be used to analyze immunospecificbinding and cross-reactivity of the antibodies of the invention include,but are not limited to, competitive and non-competitive assay systemsusing techniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).

Antibodies of the invention may also be assayed using any surfaceplasmon resonance based assays known in the art for characterizing thekinetic parameters of the interaction of the antibody with FcγRIIB AnySPR instrument commercially available including, but not limited to,BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); IAsysinstruments available from Affinity Sensors (Franklin, Mass.); IBISsystem available from Windsor Scientific Limited (Berks, UK), SPR-CELLIAsystems available from Nippon Laser and Electronics Lab (Hokkaido,Japan), and SPR Detector Spreeta available from Texas Instruments(Dallas, Tex.) can be used in the instant invention. For a review ofSPR-based technology see Mullet et al., 2000, Methods 22: 77-91; Dong etal., 2002, Review in Mol. Biotech., 82: 303-23; Fivash et al., 1998,Current Opinion in Biotechnology 9: 97-101; Rich et al., 2000, CurrentOpinion in Biotechnology 11: 54-61; all of which are incorporated hereinby reference in their entirety. Additionally, any of the SPR instrumentsand SPR based methods for measuring protein-protein interactionsdescribed in U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215;6,268,125 are contemplated in the methods of the invention, all of whichare incorporated herein by reference in their entirety.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution in real time. SPR is based on measuringthe change in refractive index of the solvent near the surface thatoccurs upon complex formation or dissociation. The surface onto whichthe immobilization occur is the sensor chip, which is at the heart ofthe SPR technology; it consists of a glass surface coated with a thinlayer of gold and forms the basis for a range of specialized surfacesdesigned to optimize the binding of a molecule to the surface. A varietyof sensor chips are commercially available especially from the companieslisted supra, all of which may be used in the methods of the invention.Examples of sensor chips include those available from BIAcore AB, Inc.,e.g., Sensor Chip CMS, SA, NTA, and HPA. A molecule of the invention maybe immobilized onto the surface of a sensor chip using any of theimmobilization methods and chemistries known in the art, including butnot limited to, direct covalent coupling via amine groups, directcovalent coupling via sulfhydryl groups, biotin attachment to avidincoated surface, aldehyde coupling to carbohydrate groups, and attachmentthrough the histidine tag with NTA chips.

The binding specificity of the antibodies of the invention may beevaluated by any method known in the art for determining binding-pairinteractions, including, but not limited to ELISA, western blot, surfaceplasmon resonance (e.g., BIAcore) and radioimmunoassay. Any method knownin the art for assessing binding specificity may be used to identifyantibodies of the invention that exhibit a Kd of greater than 0.001 nMbut not greater than 5 nM, not greater than 10 nM, not greater than 15nM, not greater than 20 nM, not greater than 25 nM, not greater than 30nM, not greater than 35 nM, not greater than 40 nM, not greater than 45nM, or not greater than 50 nM as determined by BIAcore assay.

The present invention also provides for antibodies of the invention, orfragments thereof, that have a high binding affinity for the antigen ofinterest. In a specific embodiment, an immunospecific polypeptide of thepresent invention or fragment thereof has an association rate constantor k_(on) rate (antibody (Ab)+antigen (Ag) Ab-Ag) of at least 10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹ s⁻¹ at least 10⁶ M⁻¹ s⁻¹ at least 5×10⁶ M⁻¹ s⁻¹at least 10⁷ M⁻¹ s⁻¹ at least 5×10⁷ M⁻¹ s⁻¹, or at least 10⁸ M⁻¹ s⁻¹. Ina preferred embodiment, an antibody of the present invention or fragmentthereof has a km, of at least 2×10⁵ M⁻¹ s⁻¹, at least 5×10⁵ M⁻¹ s⁻¹, atleast 10⁶ M¹ s⁻¹, at least 5×10⁶ M⁻¹ s⁻¹, at least 10⁷M M⁻¹ s⁻¹, atleast 5×10⁷ M⁻¹ s⁻¹, or at least 10⁸ M⁻¹ s⁻¹.

In another embodiment, an antibody of the present invention or fragmentthereof has a k_(off) rate (antibody (Ab)+antigen (Ag) Ab-Ag) of lessthan 10⁻¹ s⁻¹, less than 5×10⁻¹ s⁻¹, less than 10⁻² s⁻¹, less than5×10⁻² s⁻¹, less than 10⁻³ s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁻⁴s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹,less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹. In a preferredembodiment, an antibody of the present invention or fragment thereof hasa k_(off) of less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵s⁻¹, less than 10⁻⁶ s^(−i), less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹,less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, lessthan 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In still other embodiments, an antibody of the present invention orfragment thereof has an affinity constant or K_(a) (k_(on)/k_(off)) ofat least 10²M⁻¹, at least 5×10² M⁻¹, at least 10³M⁻¹, at least 5×10³M⁻¹, at least 10⁴ M⁻¹, at least 5×10⁴ M⁻¹, at least 10⁵M⁻¹, at least5×10⁵ M⁻¹, at least 10⁶M⁻¹, at least 5×10⁶ M⁻¹, at least 10⁷ M⁻¹, atleast 5×10⁷ M⁻¹, at least 10⁸M⁻¹, at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹,at least 5×10⁹ M⁻¹, at least 10¹⁰M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 5×10¹² M⁻¹, atleast 10¹³ M⁻¹, at least 5×10¹³M⁻¹, at least 10¹⁴ M⁻¹, at least 5×10¹⁴M⁻¹, at least 10¹⁵ M⁻¹, or at least 5×10¹⁵ M⁻¹. In yet anotherembodiment, an antibody of the present invention or fragment thereof hasa dissociation constant or K_(a) (k_(on)/k_(off)) of less than 10⁻² M,less than 5×10² M, less than 10⁻³M, less than 5×10⁻³ M, less than 10⁻⁴M, less than 5×10⁻⁴ M, less than 10⁻⁵M, less than 5×10⁻⁵ M, less than10⁻⁶ M, less than 5×10⁻⁶ M, less than 10⁻⁷ M, less than 5×10⁻⁷ M, lessthan 10⁻⁸M, less than 5×10⁻⁸M, less than 10⁻⁹M, less than 5×10⁻⁹M, lessthan 10⁻¹⁰ M, less than 5×10⁻¹° M, less than 10⁻¹¹ M, less than 5×10⁻¹¹M, less than 10⁻¹² M, less than 5×10⁻¹² M, less than 10⁻¹³ M, less than5×10⁻¹³ M, less than 10⁻¹⁴ M, less than 5×10⁻¹⁴ M, less than 10⁻¹⁵M, orless than 5×10⁻¹⁵ M.

The invention encompasses characterization of the antibodies produced bythe methods of the invention using certain characterization assays foridentifying the function of the antibodies of the invention,particularly the activity to modulate FcγRIIB signaling. For example,characterization assays of the invention can measure phosphorylation oftyrosine residues in the ITIM motif of FcγRIIB, or measure theinhibition of B cell receptor-generated calcium mobilization. Thecharacterization assays of the invention can be cell-based or cell-freeassays.

It has been well established in the art that in mast cells coaggregationof FcγRIIB with the high affinity IgE receptor, FcεRI, leads toinhibition of antigen-induced degranulation, calcium mobilization, andcytokine production (Metcalfe D. D. et al. 1997, Physiol. Rev. 77:1033;Long E. O. 1999 Annu Rev. Immunol. 17: 875). The molecular details ofthis signaling pathway have been recently elucidated (Ott V. L., 2002,J. Immunol. 162(9):4430-9). Once coaggregated with FcεRI, FcγRIIB israpidly phosphorylated on tyrosine in its ITIM motif, and then recruitsSrc Homology-2 containing inositol-5-phosphatase (SHIP), an SH2domain-containing inosital polyphosphate 5-phosphatase, which is in turnphosphorylated and associates with Shc and p62^(dok) (p62^(dok) is theprototype of a family of adaptor molecules, which includes signalingdomains such as an aminoterminal pleckstrin homology domain (PH domain),a PTB domain, and a carboxy terminal region containing PXXP motifs andnumerous phosphorylation sites (Carpino et al., 1997 Cell, 88: 197;Yamanshi et al., 1997, Cell, 88:205)).

The invention encompasses characterizing the anti-FcγRIIB antibodies ofthe invention in modulating one or more IgE-mediated responses.Preferably, cells lines co-expressing the high affinity receptor for IgEand the low affinity receptor for FcγRIIB will be used in characterizingthe anti-FcγRIIB antibodies of the invention in modulating IgE-mediatedresponses. In a specific embodiment, cells from a rat basophilicleukemia cell line (RBL-H23; Barsumian E. L. et al. 1981 Eur. J.Immunol. 11:317, which is incorporated herein by reference in itsentirety) transfected with full length human FcγRIIB will be used in themethods of the invention. RBL-2H3 is a well characterized rat cell linethat has been used extensively to study the signaling mechanismsfollowing IgE-mediated cell activation. When expressed in RBL-2H3 cellsand coaggregated with FcεRI, FcγRIIB inhibits FcεRI-induced calciummobilization, degranulation, and cytokine production (Malbec et al.,1998, J. Immunol. 160:1647; Daeron et al., 1995 J. Clin. Invest. 95:577;Ott et al., 2002 J. of Immunol. 168:4430-4439).

In some embodiments, the invention encompasses characterizing theanti-FcγRIIB antibodies of the invention for inhibition of FcεRI inducedmast cell activation. For example, cells from a rat basophilic leukemiacell line (RBL-H23; Barsumian E. L. et al. 1981 Eur. J. Immunol. 11:317)that have been transfected with FcγRIIB are sensitized with IgE andstimulated either with F(ab′)₂ fragments of rabbit anti-mouse IgG, toaggregate FcεRI alone, or with whole rabbit anti-mouse IgG tocoaggregate FcγRIIB and FcεRI. In this system, indirect modulation ofdown stream signaling molecules can be assayed upon addition ofantibodies of the invention to the sensitized and stimulated cells. Forexample, tyrosine phosphorylation of FcγRIIB and recruitment andphosphorylation of SHIP, activation of MAP kinase family members,including but not limited to Erk1, Erk2, JNK, or p38; and tyrosinephosphorylation of p62^(dok) and its association with SHIP and RasGAPcan be assayed.

One exemplary assay for determining the inhibition of FcεRI induced mastcell activation by the antibodies of the invention can comprise of thefollowing: transfecting RBL-H23 cells with human FcγRIIB; sensitizingthe RBL-H23 cells with IgE; stimulating RBL-H23 cells with eitherF(ab′)₂ of rabbit anti-mouse IgG (to aggregate FcεRI alone and elicitFcεRI-mediated signaling, as a control), or stimulating RBL-H23 cellswith whole rabbit anti-mouse IgG to (to coaggregate FcγRIIB and FcεRI,resulting in inhibition of FcεRI-mediated signaling). Cells that havebeen stimulated with whole rabbit anti-mouse IgG antibodies can befurther pre-incubated with the antibodies of the invention. MeasuringFcεRI-dependent activity of cells that have been pre-incubated with theantibodies of the invention and cells that have not been pre-incubatedwith the antibodies of the invention, and comparing levels ofFcεRI-dependent activity in these cells, would indicate a modulation ofFcεRI-dependent activity by the antibodies of the invention.

The exemplary assay described above can be for example, used to identifyantibodies that block ligand (IgG) binding to FcγRIIB receptor andantagonize FcγRIIB-mediated inhibition of FcεRI signaling by preventingcoaggregating of FcγRIIB and FcεRI. This assay likewise identifiesantibodies that enhance coaggregation of FcγRIIB and FcεRI and agonizeFcγRIIB-mediated inhibition of FcεRI signaling by promotingcoaggregating of FcγRIIB and FcεRI.

In a preferred embodiment, FcεRI-dependent activity is at least one ormore of the following: modulation of downstream signaling molecules(e.g., modulation of phosphorylation state of FcγRIIB, modulation ofSHIP recruitment, modulation of MAP Kinase activity, modulation ofphosphorylation state of SHIP, modulation of SHIP and Shc associationSHIP and Shc, modulation of the phosphorylation state of p62^(dok),modulation of p62^(dok) and SHIP association, modulation of p62^(dok)and RasGAP association, modulation of calcium mobilization, modulationof degranulation, and modulation of cytokine production). In yet anotherpreferred embodiment, FcεRI-dependent activity is serotonin releaseand/or extracellular Ca⁺⁺ influx and/or IgE dependent mast cellactivation. It is known to one skilled in the art that coaggregation ofFcγRIIB and FcεRI stimulates FcγRIIB tyrosine phosphorylation,stimulates recruitment of SHIP, stimulates SHIP tyrosine phosphorylationand association with Shc, and inhibits activation of MAP kinase familymembers including, but not limited to, Erk1, Erk2, JNK, p38. It is alsoknown to those skilled in the art that coaggregation of FcγRIIB andFcεRI stimulates enhanced tyrosine phosphorylation of p62^(dok) and itsassociation with SHIP and RasGAP.

In some embodiments, the anti-FcγRIIB antibodies of the invention arecharacterized for their ability to modulate an IgE-mediated response bymonitoring and/or measuring degranulation of mast cells or basophils,preferably in a cell-based assay. Preferably, mast cells or basophilsfor use in such assays have been engineered to contain human FcγRIIBusing standard recombinant methods known to one skilled in the art. In aspecific embodiment the anti-FcγRIIB antibodies of the invention arecharacterized for their ability to modulate an IgE-mediated response ina cell-based β-hexosaminidase (enzyme contained in the granules) releaseassay. β-hexosaminidase release from mast cells and basophils is aprimary event in acute allergic and inflammatory condition (Aketani etal., 2001 Immunol. Lett. 75: 185-9; Aketani et al., 2000 Anal. Chem. 72:2653-8). Release of other inflammatory mediators including but notlimited to serotonin and histamine may be assayed to measure anIgE-mediated response in accordance with the methods of the invention.Although not intending to be bound by a particular mechanism of action,release of granules such as those containing β-hexosaminidase from mastcells and basophils is an intracellular calcium concentration dependentprocess that is initiated by the cross-linking of FcγRls withmultivalent antigen.

One exemplary assay for characterizing the anti-FcγRIIB antibodies ofthe invention in mediating an IgE-mediated response is aβ-hexosaminidase release assay comprising the following: transfectingRBL-H23 cells with human FcγRIIB; sensitizing the cells with mouse IgEalone or with mouse IgE and an anti-FcγRIIB antibody of the invention;stimulating the cells with various concentrations of goat anti-mouseF(ab)₂, preferably in a range from 0.03 μg/mL to 30 μg/mL for about 1hour; collecting the supernatant; lysing the cells; and measuring theβ-hexosaminidase activity released in the supernatant by a colorometricassay, e.g., using p-nitrophenyl N-acetyl-β-D-glucosaminide. Thereleased β-hexosaminidase activity is expressed as a percentage of thereleased activity to the total activity. The released β-hexosaminidaseactivity will be measured and compared in cells treated with antigenalone; IgE alone; IgE and an anti-FcγRIIB antibody of the invention.Although not intending to be bound by a particular mechanism of action,once cells are sensitized with mouse IgE alone and challenged withF(ab)₂ fragments of a polyclonal goat anti-mouse IgG, aggregation andcross linking of FcγRI occurs since the polyclonal antibody recognizesthe light chain of the murine IgE bound to the FcεRI; which in turnleads to mast cell activation and degranulation. On the other hand, whencells are sensitized with mouse IgE and an anti-FcγRIIB antibody of theinvention and challenged with F(ab)₂ fragments of a polyclonal goatanti-mouse IgG; cross linking of FcγRI and FcγRIIB occurs, resulting ininhibition of FcγRI induced degranulation. In either case, goatanti-mouse F(ab)₂ induces a dose-dependent β-hexoaminidase release. Insome embodiments, the anti-FcγRIIB antibodies bound to the FcγRIIBreceptor and cross linked to FcγRI do not affect the activation of theinhibitory pathway, i.e., there is no alteration in the level ofdegranulation in the presence of an anti-FcγRIIB antibody. In otherembodiments, the anti-FcγRIIB antibodies mediate a stronger activationof the inhibitory receptor, FcγRIIB, when bound by the anti-FcγRIIBantibody, allowing effective cross linking to FcγRI and activation ofthe inhibitory pathway of homo-aggregated FcγRIIB

The invention also encompasses characterizing the effect of theanti-FcγRIIB antibodies of the invention on IgE-mediated cell responseusing calcium mobilization assays using methodologies known to oneskilled in the art. An exemplary calcium mobilization assay may comprisethe following: priming basophils or mast cells with IgE; incubating thecells with a calcium indicator, e.g., Fura 2; stimulating cells asdescribed supra; and monitoring and/or quantitating intracellularcalcium concentration for example by using flow cytometry. The inventionencompasses monitoring and/or quantitating intracellular calciumconcentration by any method known to one skilled in the art see, e.g.,Immunology Letters, 2001, 75:185-9; British J. of Pharm, 2002,136:837-45; J. of Immunology, 168:4430-9 and J. of Cell Biol.,153(2):339-49; all of which are incorporated herein by reference.

In preferred embodiments, anti-FcγRIIB antibodies of the inventioninhibit IgE-mediated cell activation. In other embodiments, theanti-FcγRIIB antibodies of the invention block the inhibitory pathwaysregulated by FcγRIIB or block the ligand binding site on FcγRIIB andthus enhance immune response.

The ability to study human mast cells has been limited by the absence ofsuitable long term human mast cell cultures. Recently two novel stemcell factor dependent human mast cell lines, designated LAD 1 and LAD2,were established from bone marrow aspirates from a patient with mastcell sarcoma/leukemia (Kirshenbaum et al., 2003, Leukemia research,27:677-82, which is incorporated herein by reference in its entirety.).Both cell lines have been described to express FcεRI and several humanmast cell markers. The invention encompasses using LAD 1 and 2 cells inthe methods of the invention for assessing the effect of the antibodiesof the invention on IgE-mediated responses. In a specific embodiment,cell-based β-hexosaminidase release assays such as those described supramay be used in LAD cells to determine any modulation of the IgE-mediatedresponse by the anti-FcγRIIB antibodies of the invention. In anexemplary assay, human mast cells, e.g., LAD 1, are primed with chimerichuman IgE anti-nitrophenol (NP) and challenged with BSA-NP, thepolyvalent antigen, and cell degranulation is monitored by measuring theβ-hexosaminidase released in the supernatant (Kirshenbaum et al., 2003,Leukemia research, 27:677-682, which is incorporated herein by referencein its entirety).

In some embodiments, if human mast cells have a low expression ofendogenous FcγRIIB, as determined using standard methods known in theart, e.g., FACS staining, it may be difficult to monitor and/or detectdifferences in the activation of the inhibitory pathway mediated by theanti-FcγRIIB antibodies of the invention. The invention thus encompassesalternative methods, whereby the FcγRIIB expression may be upregulatedusing cytokines and particular growth conditions. FcγRIIB has beendescribed to be highly up-regulated in human monocyte cell lines, e.g.,THP1 and U937, (Tridandapani et al., 2002, J. Biol. Chem., 277(7):5082-5089) and in primary human monocytes (Pricop et al., 2001, J. ofImmunol., 166: 531-537) by IL4. Differentiation of U937 cells withdibutyryl cyclic AMP has been described to increase expression of FcγRII(Cameron et al., 2002 Immunology Letters 83, 171-179). Thus theendogenous FcγRIIB expression in human mast cells for use in the methodsof the invention may be up-regulated using cytokines, e.g., IL-4, IL-13,in order to enhance sensitivity of detection.

The invention also encompasses characterizing the anti-FcγRIIBantibodies of the invention for inhibition of B-cell receptor(BCR)-mediated signaling. BCR-mediated signaling can include at leastone or more down stream biological responses, such as activation andproliferation of B cells, antibody production, etc. Coaggregation ofFcγRIIB and BCR leads to inhibition of cell cycle progression andcellular survival. Further, coaggregation of FcγRIIB and BCR leads toinhibition of BCR-mediated signaling.

Specifically, BCR-mediated signaling comprises at least one or more ofthe following: modulation of down stream signaling molecules (e.g.,phosphorylation state of FcγRIIB, SHIP recruitment, localization of Btkand/or PLCγ, MAP kinase activity, recruitment of Akt (anti-apoptoticsignal), calcium mobilization, cell cycle progression, and cellproliferation).

Although numerous effector functions of FcγRIIB-mediated inhibition ofBCR signaling are mediated through SHIP, recently it has beendemonstrated that lipopolysaccharide (LPS)-activated B cells from SHIPdeficient mice exhibit significant FcγRIIB-mediated inhibition ofcalcium mobilization, Ins(1,4,5)P₃ production, and Erk and Aktphosphorylation (Brauweiler A. et al., 2001, Journal of Immunology,167(1): 204-211). Accordingly, ex vivo B cells from SHIP deficient micecan be used to characterize the antibodies of the invention. Oneexemplary assay for determining FcγRIIB-mediated inhibition of BCRsignaling by the antibodies of the invention can comprise the following:isolating splenic B cells from SHIP deficient mice, activating saidcells with lipopolysachharide, and stimulating said cells with eitherF(ab′)₂ anti-IgM to aggregate BCR or with anti-IgM to coagregate BCRwith FcγRIIB Cells that have been stimulated with intact anti-IgM tocoaggregate BCR with FcγRIIB can be further pre-incubated with theantibodies of the invention. FcγRIIB-dependent activity of cells can bemeasured by standard techniques known in the art. Comparing the level ofFcγRIIB-dependent activity in cells that have been pre-incubated withthe antibodies of the invention and cells that have not beenpre-incubated, and comparing the levels would indicate a modulation ofFcγRIIB-dependent activity by the antibodies of the invention.

Measuring FcγRIIB-dependent activity can include, for example, measuringintracellular calcium mobilization by flow cytometry, measuringphosphorylation of Akt and/or Erk, measuring BCR-mediated accumulationof PI(3,4,5)P₃, or measuring FcγRIIB-mediated proliferation B cells.

The assays can be used, for example, to identify antibodies thatmodulate FcγRIIB-mediated inhibition of BCR signaling by blocking theligand (IgG) binding site to FcγRIIB receptor and antagonizingFcγRIIB-mediated inhibition of BCR signaling by preventing coaggregationof FcγRIIB and BCR. The assays can also be used to identify antibodiesthat enhance coaggregation of FcγRIIB and BCR and agonizeFcγRIIB-mediated inhibition of BCR signaling.

The invention relates to characterizing the anti-FcγRIIB antibodies ofthe invention for FcγRII-mediated signaling in humanmonocytes/macrophages. Coaggregation of FcγRIIB with a receptor bearingthe immunoreceptor tyrosine-based activation motif (ITAM) acts todown-regulate FcγR-mediated phagocytosis using SHIP as its effector(Tridandapani et al. 2002, J. Biol. Chem. 277(7):5082-9). Coaggregationof FcγRIIA with FcγRIIB results in rapid phosphorylation of the tyrosineresidue on FcγRIIB's ITIM motif, leading to an enhancement inphosphorylation of SHIP, association of SHIP with Shc, andphosphorylation of proteins having the molecular weight of 120 and 60-65kDa. In addition, coaggregation of FcγRIIA with FcγRIIB results indown-regulation of phosphorylation of Akt, which is a serine-threoninekinase that is involved in cellular regulation and serves to suppressapoptosis.

The invention further encompasses characterizing the anti-FcγRIIBantibodies of the invention for their inhibition of FcγR-mediatedphagocytosis in human monocytes/macrophages. For example, cells from ahuman monocytic cell line, THP-1 can be stimulated either with Fabfragments of mouse monoclonal antibody IV.3 against FcγRII and goatanti-mouse antibody (to aggregate FcγRIIA alone), or with whole IV.3mouse monoclonal antibody and goat anti-mouse antibody (to coaggregateFcγRIIA and FcγRIIB) In this system, modulation of down stream signalingmolecules, such as tyrosine phosphorylation of FcγRIIB, phosphorylationof SHIP, association of SHIP with Shc, phosphorylation of Akt, andphosphorylation of proteins having the molecular weight of 120 and 60-65kDa can be assayed upon addition of antibodies of the invention to thestimulated cells. In addition, FcγRIIB-dependent phagocytic efficiencyof the monocyte cell line can be directly measured in the presence andabsence of the antibodies of the invention.

Another exemplary assay for determining inhibition of FcγR-mediatedphagocytosis in human monocytes/macrophages by the antibodies of theinvention can comprise the following: stimulating THP-1 cells witheither Fab of IV.3 mouse anti-FcγRII antibody and goat anti-mouseantibody (to aggregate FcγRIIA alone and elicit FcγRIIA-mediatedsignaling); or with mouse anti-FcγRII antibody and goat anti-mouseantibody (to coaggregate FcγRIIA and FcγRIIB and inhibitingFcγRIIA-mediated signaling. Cells that have been stimulated with mouseanti-FcγRII antibody and goat anti-mouse antibody can be furtherpre-incubated with the antibodies of the invention. MeasuringFcγRIIA-dependent activity of stimulated cells that have beenpre-incubated with antibodies of the invention and cells that have notbeen pre-incubated with the antibodies of the invention and comparinglevels of FcγRIIA-dependent activity in these cells would indicate amodulation of FcγRIIA-dependent activity by the antibodies of theinvention.

The exemplary assay described can be used for example, to identifyantibodies that block ligand binding of FcγRIIB receptor and antagonizeFcγRIIB-mediated inhibition of FcγRIIA signaling by preventingcoaggregation of FcγRIIB and FcγRIIA. This assay likewise identifiesantibodies that enhance coaggregation of FcγRIIB and FcγRIIA and agonizeFcγRIIB-mediated inhibition of FcγRIIA signaling.

In another embodiment of the invention, the invention relates tocharacterizing the function of the antibodies of the invention bymeasuring the ability of THP-1 cells to phagocytose fluoresceinatedIgG-opsonized sheep red blood cells (SRBC) by methods previouslydescribed (Tridandapani et al., 2000, J. Biol. Chem. 275: 20480-7). Forexample, an exemplary assay for measuring phagocytosis comprises of:treating THP-1 cells with the antibodies of the invention or with acontrol antibody that does not bind to FcγRII, comparing the activitylevels of said cells, wherein a difference in the activities of thecells (e.g., rosetting activity (the number of THP-1 cells bindingIgG-coated SRBC), adherence activity (the total number of SRBC bound toTHP-1 cells), and phagocytic rate) would indicate a modulation ofFcγRIIA-dependent activity by the antibodies of the invention. Thisassay can be used to identify, for example, antibodies that block ligandbinding of FcγRIIB receptor and antagonize FcγRIIB-mediated inhibitionof phagocytosis. This assay can also identify antibodies that enhanceFcγRIIB-mediated inhibition of FcγRIIA signaling.

In a preferred embodiment, the antibodies of the invention modulateFcγRIIB-dependent activity in human monocytes/macrophages in at leastone or more of the following ways: modulation of downstream signalingmolecules (e.g., modulation of phosphorylation state of FcγRIIB,modulation of SHIP phosphorylation, modulation of SHIP and Shcassociation, modulation of phosphorylation of Akt, modulation ofphosphorylation of additional proteins around 120 and 60-65 kDa) andmodulation of phagocytosis.

The invention encompasses characterization of the antibodies of theinvention using assays known to those skilled in the art for identifyingthe effect of the antibodies on effector cell function of therapeuticantibodies, e.g., their ability to enhance tumor-specific ADCC activityof therapeutic antibodies. Therapeutic antibodies that may be used inaccordance with the methods of the invention include but are not limitedto anti-tumor antibodies, anti-viral antibodies, anti-microbialantibodies (e.g., bacterial and unicellular parasites), examples ofwhich are disclosed herein (Section 5.4.6). In particular, the inventionencompasses characterizing the antibodies of the invention for theireffect on FcγR-mediated effector cell function of therapeuticantibodies, e.g., tumor-specific monoclonal antibodies. Examples ofeffector cell functions that can be assayed in accordance with theinvention, include but are not limited to, antibody-dependentcell-mediated cytotoxicity, phagocytosis, opsonization,opsonophagocytosis, C1q binding, and complement dependent cell mediatedcytotoxicity. Any cell-based or cell free assay known to those skilledin the art for determining effector cell function activity can be used(For effector cell assays, see Perussia et al., 2000, Methods Mol. Biol.121: 179-92; Baggiolini et al., 1998 Experientia, 44(10): 841-8; Lehmannet al., 2000 J. Immunol. Methods, 243(1-2): 229-42; Brown E J. 1994,Methods Cell Biol., 45: 147-64; Munn et al., 1990 J. Exp. Med., 172:231-237, Abdul-Majid et al., 2002 Scand. J. Immunol. 55: 70-81; Ding etal., 1998, Immunity 8:403-411, each of which is incorporated byreference herein in its entirety).

Antibodies of the invention can be assayed for their effect onFcγR-mediated ADCC activity of therapeutic antibodies in effector cells,e.g., natural killer cells, using any of the standard methods known tothose skilled in the art (See e.g., Perussia et al., 2000, Methods Mol.Biol. 121: 179-92). “Antibody-dependent cell-mediated cytotoxicity” and“ADCC” as used herein carry their ordinary and customary meaning in theart and refer to an in vitro cell-mediated reaction in which nonspecificcytotoxic cells that express FcγRs (e.g., monocytic cells such asNatural Killer (NK) cells and macrophages) recognize bound antibody on atarget cell and subsequently cause lysis of the target cell. Inprinciple, any effector cell with an activating FcγR can be triggered tomediate ADCC. The primary cells for mediating ADCC are NK cells whichexpress only FcγRIII, whereas monocytes, depending on their state ofactivation, localization, or differentiation, can express FcγRI, FcγRII,and FcγRIII. For a review of FcγR expression on hematopoietic cells see,e.g., Ravetch et al., 1991, Annu. Rev. Immunol., 9:457-92, which isincorporated herein by reference in its entirety.

Effector cells are leukocytes which express one or more FcγRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Effector cells that may beused in the methods of the invention include but are not limited toperipheral blood mononuclear cells (PBMC), natural killer (NK) cells,monocytes, and neutrophils; with PBMCs and NK cells being preferred. Theeffector cells may be isolated from a native source thereof, e.g., fromblood or PBMCs as described herein. Preferably, the effector cells usedin the ADCC assays of the invention are peripheral blood mononuclearcells (PBMC) that are preferably purified from normal human blood, usingstandard methods known to one skilled in the art, e.g., usingFicoll-Paque density gradient centrifugation. For example, PBMCs may beisolated by layering whole blood onto Ficoll-Hypaque and spinning thecells at 500 g, at room temperature for 30 minutes. The leukocyte layercan be harvested as effector cells. Other effector cells that may beused in the ADCC assays of the invention include but are not limited tomonocyte-derived macrophages (MDMs). MDMs that are used as effectorcells in the methods of the invention, are preferably obtained as frozenstocks or used fresh, (e.g., from Advanced Biotechnologies, MD). In mostpreferred embodiments, elutriated human monocytes are used as effectorcells in the methods of the invention. Elutriated human monocytesexpress activating receptors, FcγRIIIA and FcγRIIA and the inhibitoryreceptor, FcγRIIB Human monocytes are commercially available and may beobtained as frozen stocks, thawed in basal medium containing 10% humanAB serum or in basal medium with human serum containing cytokines.Levels of expression of FcγRs in the cells may be directly determined;e.g. using FACS analysis. Alternatively, cells may also be allowed tomature to macrophages in culture. The level of FcγRIIB expression may beincreased in macrophages. Antibodies that may be used in determining theexpression level of FcγRs include but are not limited to anti-humanFcγRIIA antibodies, e.g., IV.3-FITC; anti-FcγRI antibodies, e.g., 32.2FITC; and anti-FcγRIIIA antibodies, e.g., 3G8-PE.

Target cells used in the ADCC assays of the invention include, but arenot limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al., 1976, Cancer Res.33-41); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Rajicells with ATCC accession number CCL-86 (see, e.g., Epstein et al.,1965, J. Natl. Cancer Inst. 34: 231-240), Daudi cells with ATCCaccession number CCL-213 (see, e.g., Klein et al., 1968, Cancer Res. 28:1300-10); ovarian carcinoma cell lines, e.g., OVCAR-3 with ATCCaccession number HTB-161 (see, e.g., Hamilton, Young et al., 1983),SK-OV-3, PA-1, CAOV3, OV-90, and IGROV-1 (available from the NCIrepository Benard et al., 1985, Cancer Research, 45:4970-9; which isincorporated herein by reference in its entirety. The target cells mustbe recognized by the antigen binding site of the antibody to be assayed.The target cells for use in the methods of the invention may have low,medium, or high expression level of a cancer antigen. The expressionlevels of the cancer antigen may be determined using common methodsknown to one skilled in the art, e.g., FACS analysis. For example, theinvention encompasses the use of ovarian cancer cells such as IGROV-1,wherein Her2/neu is expressed at different levels, or OV-CAR-3 (ATCCAssession Number HTB-161; characterized by a lower expression ofHer2/neu than SK-BR-3, the breast carcinoma cell line). Other ovariancarcinoma cell lines that may be used as target cells in the methods ofthe invention include OVCAR-8 (Hamilton et al., 1983, Cancer Res.43:5379-89, which is incorporated herein by reference in its entirety);SK-OV-3 (ATCC Accession Number HTB-77); Caov-3 (ATCC Accession NumberHTB-75); PA-1 (ATCC Accession Number CRL-1572); OV-90 (ATCC AccessionNumber CRL-11732); and OVCAR-4. Other breast cancer cell lines that maybe used in the methods of the invention include BT-549 (ATCC AccessionNumber HTB-122), MCF7 (ATCC Accession Number HTB-22), and Hs578T (ATCCAccession Number HTB-126), all of which are available from the NCIrepository and ATCC and incorporated herein by reference. Other celllines that may be used in the methods of the invention include but arenot limited to CCRF-CEM (leukemia); HL-60 (TB, leukemia); MOLT-4(leukemia); RPMI-8226 (leukemia); SR (leukemia); A549 (Non-small celllung); EKVX (Non-small cell lung); HOP-62 (Non-small cell lung); HOP-92(Non-small cell lung); NC1-H226 (Non-small cell lung); NC1-H23(Non-small cell lung); NC1-H322M (Non-small cell lung); NC1-H460(Non-small cell lung); NC1-H522 (Non-small cell lung); COLO 205 (Colon);HCC-2998 (Colon); HCT-116 (Colon); HCT-15 (Colon); HT29 (Colon); KM12(Colon); SW-620 (Colon); SF-268 (CNS); SF-295 (CNS); SF-539 (CNS);SNB-19 (CNS); SNB-75 (CNS); U251 (CNS); LOX 1MV1 (Melanoma); MALME-3M(Melanoma); M14 (Melanoma); SK-MEL-2 (Melanoma); SK-MEL-28 (Melanoma);SK-MEL-5 (Melanoma); UACC-257 (Melanoma); UACC-62 (Melanoma); IGR-OV1(Ovarian); OVCAR-3, 4, 5, 8 (Ovarian); SK-OV-3 (Ovarian); 786-0 (Renal);A498 (Renal); ACHN (Renal); CAK1-1 (Renal); SN12C(Renal); TK-10 (Renal);UO-31 (Renal); PC-3C (Prostate); DU-145 (Prostate); NC1/ADR-RES(Breast); MDA-MB-231/ATCC (Breast); MDA-MB-435 (Breast); DMS 114(Small-cell lung); and SHP-77 (Small-cell lung); all of which areavailable from the NC1 and incorporated herein by reference.

An exemplary assay for determining the effect of the antibodies of theinvention on the ADCC activity of therapeutic antibodies is based on a⁵¹Cr release assay comprising of: labeling target cells with[⁵¹Cr]Na₂CrO₄ (this cell-membrane permeable molecule is commonly usedfor labeling since it binds cytoplasmic proteins and althoughspontaneously released from the cells with slow kinetics, it is releasedmassively following target cell lysis); preferably, the target cellsexpress one or more tumor antigens, osponizing the target cells with oneor more antibodies that immunospecifically bind the tumor antigensexpressed on the cell surface of the target cells, in the presence andabsence of an antibody of the invention, e.g., 8B5.3.4, combining theopsonized radiolabeled target cells with effector cells in a microtitreplate at an appropriate ratio of target cells to effector cells;incubating the mixture of cells preferably for 16-18 hours, preferablyat 37° C.; collecting supernatants; and analyzing the radioactivity inthe supernatant samples. The cytotoxicity of the therapeutic antibodiesin the presence and absence of the antibodies of the invention can thenbe determined, for example using the following formula: Percent specificlysis=(Experimental lysis-antibody-independent lysis/maximallysis−antibody independent lysis)×100%. A graph can be generated byvarying either the target: effector cell ratio or antibodyconcentration.

In yet another embodiment, the antibodies of the invention arecharacterized for antibody-dependent cell-mediated cytotoxicity (ADCC)in accordance with the method described earlier, see, e.g., Ding et al.,Immunity, 1998, 8:403-11; which is incorporated herein by reference inits entirety.

In some embodiments, the invention encompasses characterizing thefunction of the antibodies of the invention in enhancing ADCC activityof therapeutic antibodies in an in vitro based assay and/or in an animalmodel.

In a specific embodiment, the invention encompasses determining thefunction of the antibodies of the invention in enhancing tumor specificADCC using an ovarian cancer model and/or breast cancer model.

Preferably, the ADCC assays of the invention are done using more thanone cancer cell line, characterized by the expression of at least onecancer antigen, wherein the expression level of the cancer antigen isvaried among the cancer cell lines used. Although not intending to bebound by a particular mechanism of action, performing ADCC assays inmore than one cell line wherein the expression level of the cancerantigen is varied, will allow determination of stringency of tumorclearance of the antibodies of the invention. In one embodiment, theADCC assays of the invention are done using cancer cell lines withdifferent levels of expression of a cancer antigen.

In an exemplary assay, OVCAR3, an ovarian carcinoma cell line can serveas the tumor target expressing the tumor antigens, Her2/neu and TAG-72;human monocytes, that express the activating FcγRIIIA and FcγRIIA andinhibitory FcγRIIB, can be used as effectors; and tumor specific murineantibodies, ch4D5 and chCC49, can be used as the tumor specificantibodies. OVCAR-3 cells are available from ATCC (Accession NumberHTB-161). Preferably, OVCAR-3 cells are propagated in mediumsupplemented with 0.01 mg/ml bovine insulin. 5×10⁶ viable OVCAR-3 cellsmay be injected subcutaneously (s.c.) into age and weight matched nudeathymic mice with Matrigel (Becton Dickinson). The estimated weight ofthe tumor can be calculated by the formula: length-(width)/2, andpreferably does not exceed 3 grams. Anchorage-dependent tumor can beisolated after 6-8 weeks, and the cells can be dissociated by adding 1μg of Collagenase (Sigma) per gram of tumor and a 5 mg/mL RNase, passedthrough a cell strainer and nylon mesh to isolate cells. Cells can thenbe frozen for long-term storage for s.c. injection for establishment ofthe xenograft model.

Hybridomas secreting CC49 and 4D5 antibodies are available with ATCCAccession Numbers HB-9459 and CRL-3D463 and the heavy chain and lightchain nucleotide sequences are in the public domain (Murray et al., 1994Cancer 73 (35):1057-66, Yamamoto et al., 1986 Nature, 319:230-4; both ofwhich are incorporated herein by reference in their entirety).Preferably, the 4D5 and CC49 antibodies are chimerized using standardmethods known to one skilled in the art so that the human Fc sequence,e.g., human constant region of IgG1, is grafted onto the variable regionof the murine antibodies in order to provide the effector function. Thechimeric 4D5 and CC49 antibodies bind via their variable region to thetarget cell lines and via their Fc region to FcγRs expressed on humaneffector cells. CC49 is directed to TAG-72; a high molecular weightmucin that is highly expressed on many adenocarcinoma cells and ovariancarcinoma (Lottich et al., 1985 Breast Cancer Res. Treat. 6(1):49-56;Mansi et al., 1989 Int. J. Rad. Appl. Instrum B. 16(2):127-35; Colcheret al., 1991 Int. J. Rad. Appl. Instrum B. 18:395-41; all of which areincorporated herein by reference in their entirety). The 4D5 antibody isdirected to human epidermal growth factor receptor 2 (Carter et al.,1992, Proc. Natl. Acad. Sci. USA, 89: 4285-9 which is incorporatedherein by reference). Antibodies of the invention can then be utilizedto investigate the enhancement of ADCC activity of the tumor specificantibodies, by blocking the inhibitory FcγRIIB Although not intending tobe bound by a particular mechanism of action, upon activation ofeffector cells that express at least one activating FcγR, e.g., FcγRIIA,the expression of the inhibitory receptor (FcγRIIB) is enhanced and thislimits the clearance of tumors as the ADCC activity of FcγRIIA issuppressed. However, antibodies of the invention can serve as a blockingantibody, i.e., an antibody that will prevent the inhibitory signal frombeing activated and thus the activation signal, e.g., ADCC activity,will be sustained for a longer period and may result in potent tumorclearance.

Preferably, the antibodies of the invention for use in enhancement ofADCC activity have been modified to comprise at least one amino acidmodification, so that their binding to FcγR has been diminished, mostpreferably abolished. In some embodiments, the antibodies of theinvention have been modified to comprise at least one amino acidmodification which reduces the binding of the constant domain to anactivating FcγR, e.g., FcγRIIIA, FcγRIIA, as compared to a wild typeantibody of the invention while retaining maximal FcγRIIB blockingactivity. Antibodies of the invention may be modified in accordance withany method known to one skilled in the art or disclosed herein. Anyamino acid modification which is known to disrupt effector function maybe used in accordance with the methods of the invention such as thosedisclosed in U.S. Provisional Application Nos. 60/439,498 (filed Jan. 9,2003) and 60/456,041 (filed Mar. 19, 2003) and U.S. patent applicationSer. No. 10/754,922 (filed Jan. 9, 2004) and Ser. No. 10/902,588 (filedJul. 28, 2004); which are incorporated herein by reference in theirentireties. In some embodiments, antibodies of the invention aremodified so that position 265 is modified, e.g., position 265 issubstituted with alanine. In preferred embodiments, the murine constantregion of an antibody of the invention is swapped with the correspondinghuman constant region comprising a substitution of the amino acid atposition 265 with alanine, so that the effector function is abolishedwhile FcγRIIB blocking activity is maintained. A single amino acidchange at position 265 of IgG1 heavy chain has been shown tosignificantly reduce binding to FcγR based on ELISA assays, Sheilds etal., 2001, J. Biol. Chem., 276(9):6591-604; which is incorporated hereinby reference in its entirety and has resulted in tumor mass reduction.In other embodiments, antibodies of the invention are modified so thatposition 297 is modified, e.g., position 297 is substituted withglutamine, so that the N-linked glycosylation site is eliminated (see,e.g., Jefferies et al., 1995, Immunol. lett 44:111-7; Lund et al., 1996,J. Immunol., 157:4963-69; Wright et al., 1994, J. Exp. Med. 180:1087-96;White et al., 1997; J. Immunol. 158:426-35; all of which areincorporated herein by reference in their entireties. Modification atthis site has been reported to abolish all interaction with FcγRs. Inpreferred embodiments, the murine constant region of an antibody of theinvention is swapped with the corresponding human constant regioncomprising a substitution of the amino acid at position 265 and/or 297,so that the effector function is abolished while FcγRIIB blockingactivity is maintained.

An exemplary assay for determining the ADCC activity of the tumorspecific antibodies in the presence and absence of the antibodies of theinvention is a non-radioactive europium based fluorescent assay (BATDA,Perkin Elmer) and may comprise the following: labeling the targets cellswith an acteoxylmethyl ester of fluorescence-enhancing ester that formsa hydrophilic ligand (TDA) with the membrane of cells by hydrolysis ofthe esters; this complex is unable to leave the cell and is releasedonly upon lysis of the cell by the effectors; adding the labeled targetsto the effector cells in presence of anti-tumor antibodies and anantibody of the invention; incubating the mixture of the target andeffector cells a for 6 to 16 hours, preferably at 37° C. The extent ofADCC activity can be assayed by measuring the amount of ligand that isreleased and interacts with europium (DELFIA reagent; PerkinElmer). Theligand and the europium form a very stable and highly fluorescentchelate (EuTDA) and the measured fluorescence is directly proportionalto the number of cells lysed. Percent specific lysis can be calculatedusing the formula: (Experimental lysis-antibody-independentlysis/maximal lysis antibody-independent lysis×100%).

In some embodiments, if the sensitivity of the fluorescence-based ADCCassay is too low to detect ADCC activity of the therapeutic antibodies,the invention encompasses radioactive-based ADCC assays, such as ⁵¹Crrelease assay. Radioactive-based assays may be done instead of or incombination with fluorescent-based ADCC assays.

An exemplary ⁵¹Cr release assay for characterizing the antibodies of theinvention can comprise the following: labeling 1-2×10⁶ target cells suchas OVCAR-3 cells with ⁵¹Cr; opsonizing the target cells with antibodies4D5 and CC49 in the presence and absence of an antibody of the inventionand adding 5×10³ cells to 96 well plate. Preferably 4D5 and CC49 are ata concentration varying from 1-15 μg/mL; adding the opsonized targetcells to monocyte-derived macrophages (MDM) (effector cells); preferablyat a ratio varying from 10:1 to 100:1; incubating the mixture of cellsfor 16-18 hours at 37° C.; collecting supernatants; and analyzing theradioactivity in the supernatant. The cytotoxicity of 4D5 and CC49 inthe presence and absence of an antibody of the invention can then bedetermined, for example using the following formula percent specificlysis=(experimental lysis−antibody independent lysis/maximallysis−antibody independent lysis)×100%.

In some embodiments, the in vivo activity of the FcγRIIB antibodies ofthe invention is determined in xenograft human tumor models. Tumors maybe established using any of the cancer cell lines described supra. Insome embodiments, the tumors will be established with two cancer celllines, wherein the first cancer cell line is characterized by a lowexpression of a cancer antigen and a second cancer cell line, whereinthe second cancer cell line is characterized by a high expression of thesame cancer antigen. Tumor clearance may then be determined usingmethods known to one skilled in the art, using an anti-tumor antibodywhich immunospecifically binds the cancer antigen on the first andsecond cancer cell line, and an appropriate mouse model, e.g., a Balb/cnude mouse model (e.g., Jackson Laboratories, Taconic), with adoptivelytransferred human monocytes and MDMs as effector cells. Any of theantibodies described supra may then be tested in this animal model toevaluate the role of anti-FcγRIIB antibody of the invention in tumorclearance. Mice that may be used in the invention include for exampleFcγRIII−/− (where FcγRIIIA is knocked out); Fcγ−/−□ nude mice (whereFcγRI and FcγRIIIA are knocked out); or human FcγRIIB knock in mice or atransgenic knock-in mice, where mouse fcgr2 and fcgr3 loci on chromosome1 are inactivated and the mice express human FcγRIIA, human FcγRIIAhuman FcγRIIB, human FcγRIIC, human FcγRIIIA, and human FcγRIIIB

An exemplary method for testing the in vivo activity of an antibody ofthe invention may comprise the following: establishing a xenograftmurine model using a cancer cell line characterized by the expression ofa cancer antigen and determining the effect of an antibody of theinvention on an antibody specific for the cancer antigen expressed inthe cancer cell line in mediating tumor clearance. Preferably, the invivo activity is tested parallel using two cancer cell lines, whereinthe first cancer cell line is characterized by a first cancer antigenexpressed at low levels and a second cancer cell line, characterized bythe same cancer antigen expressed at a higher level relative to thefirst cancer cell line. These experiments will thus increase thestringency of the evaluation of the role of an antibody of the inventionin tumor clearance. For example, tumors may be established with theIGROV-1 cell line and the effect of an anti-FcγRIIB antibody of theinvention in tumor clearance of a Her2/neu specific antibody may beassessed. In order to establish the xenograft tumor models, 5×10⁶ viablecells, e.g., IGROV-1, SKBR3, may be injected, e.g., s.c. into mice,e.g., 8 age and weight matched female nude athymic mice using forexample Matrigel (Becton Dickinson). The estimated weight of the tumormay be determined by the formula: length×(width)/2; and preferably doesnot exceed 3 grams. Injection of IGROV-1 cells s.c. gives rise to fastgrowing tumors while the i.p. route induces a peritoneal carcinomatosiswhich kills mice in 2 months (Benard et al., 1985, Cancer Res.45:4970-9). Since the IGROV-1 cells form tumors within 5 weeks, at day 1after tumor cell injection, monocytes as effectors are co-injected i.p.along with a therapeutic antibody specific for Her2/neu, e.g., Ch4D5,and an antibody of the invention; e.g. chimeric 8B5.3.4 antibody(“ch8B5.3.4”) as described supra. Preferably, the antibodies areinjected at 4 μg each per gram of mouse body weight (mbw). The initialinjection will be followed by weekly injections of antibodies for 4-6weeks thereafter at 2 μg/wk. Human effector cells will be replenishedonce in 2 weeks. A group of mice will receive no therapeutic antibodybut will be injected with a chimeric 4D5 comprising a N297A mutation andhuman IgG1 as isotype control antibodies for the anti-tumor andanti-FcγRIIB antibodies, respectively. Mice may be placed in groups of 4and monitored three times weekly.

Table 5 below is an exemplary setup for tumor clearance studies inaccordance with the invention. As shown in Table 5, six groups of 8 miceeach will be needed for testing the role of an antibody of the inventionin tumor clearance, wherein one target and effector cell combination isused and wherein two different combinations of the antibodyconcentration are used. In group A, only tumor cells are injected; ingroup B tumor cells and monocytes are injected; in group C, tumor cells,monocytes, an anti-tumor antibody (ch4D5) are injected; in group D,tumor cells, monocytes, anti-tumor antibody, and an anti-FcγRII antibodyare injected; in group E, tumor cells, monocytes and an anti-FcγRIIBantibody are injected; in group F, tumor cells, monocytes, Ch4D5(N297Q), and human IgG1 are injected. It will be appreciated by oneskilled in the art that various antibody concentrations of variousantibody combinations may be tested in the tumor models described.Preferably, studies using a breast cancer cell line, e.g., SKBR3, iscarried out in parallel to the above-described experiment.

TABLE 5 EXEMPLARY EXPERIMENTAL SET UP IN MICE ch4D5 ch8B5.3.4 HumanMono- ch4D5 at N297Q at N297Q at IgG1 8 Tumor cytes 4 μg/gm 4 μg/gm 4μg/gm 4 μg/gm mice/ cell s.c i.p at of mbw of mbw of mbw of mbw groupday 0 day 1 day 1 i.p day 1 i.p day 1 i.p day 1 i.p A + — — — — — B + +— — — — C + + + — — — D + + + — + — E + + — — + — F + + — + — +

The endpoint of the xenograft tumor models is determined based on thesize of the tumors, weight of mice, survival time and histochemical andhistopathological examination of the cancer, using methods known to oneskilled in the art. Each of the groups of mice in Table 5 will beevaluated. Mice are preferably monitored three times a week. Criteriafor tumor growth may be abdominal distention, presence of palpable massin the peritoneal cavity. Preferably estimates of tumor weight versusdays after inoculation will be calculated. A comparison of theaforementioned criteria of mice in Group D compared to those in othergroups will define the role of an antibody of the invention inenhancement of tumor clearance. Preferably, antibody-treated animalswill be under observation for an additional 2 months after the controlgroup.

In alternative embodiments, human FcγRIIB “knock in” mice expressinghuman FcγRIIB on murine effector cells may be used in establishing thein vivo activity of the antibodies of the invention, rather thanadoptively transferring effector cells. Founder mice expressing thehuman FcγRIIB may be generated by “knocking in” the human FcγRIIB ontothe mouse FcγRIIB locus. The founders can then be back-crossed onto thenude background and will express the human FcγRIIB receptor. Theresulting murine effector cells will express endogenous activating FcγRTand FcγRIIIA and inhibitory human FcγRIIB receptors.

The in vivo activity of the antibodies of the invention may be furthertested in a xenograft murine model with human primary tumor derivedcells, such as human primary ovarian and breast carcinoma derived cells.Ascites and pleural effusion samples from cancer patients may be testedfor expression of Her2/neu, using methods known to one skilled in theart. Samples from ovarian carcinoma patients may be processed byspinning down the ascites at 6370 g for 20 minutes at 4° C., lysing thered blood cells, and washing the cells with PBS. Once the expression ofHer2/neu in tumor cells is determined, two samples, a median and a highexpressor may be selected for s.c. inoculation to establish thexenograft tumor model. The isolated tumor cells will then be injectedi.p. into mice to expand the cells. Approximately 10 mice may beinjected i.p. and each mouse ascites further passaged into two mice toobtain ascites from a total of 20 mice which can be used to inject agroup of 80 mice. Pleural effusion samples may be processed using asimilar method as ascites. The Her2/neu+tumor cells from pleuraleffusion samples may be injected into the upper right & left mammarypads of the mice.

In some embodiments, if the percentage of neoplastic cells in theascites or pleural effusion samples is low compared to other cellularsubsets, the neoplastic cells may be expanded in vitro. In otherembodiments, tumor cells may be purified using CC49 antibody(anti-TAG-72)-coated magnetic beads as described previously, see, e.g.,Barker et al., 2001, Gynecol. Oncol. 82:57, 63, which is incorporatedherein by reference in its entirety. Briefly, magnetic beads coated withCC49 antibody can be used to separate the ovarian tumor cells that willbe detached from the beads by an overnight incubation at 37° C. In someembodiments, if the tumor cells lack the TAG-72 antigen, negativedepletion using a cocktail of antibodies, such as those provided by StemCell Technologies, Inc., Canada, may be used to enrich the tumor cells.

In other embodiments, other tumors markers besides Her2/neu may be usedto separate tumor cells obtained from the ascites and pleural effusionsamples from non-tumor cells. In the case of pleural effusion or breasttissue, it has been recently reported that CD44 (an adhesion molecule),B38.1 (a breast/ovarian cancer-specific marker), CD24 (an adhesionmolecule) may be used as markers, see, e.g., Al Hajj, et al., 2003,Proc. Natl. Acad. Sci. USA 100:3983, 8; which is incorporated herein byreference in its entirety. Once tumor cells are purified they may beinjected s.c. into mice for expansion.

Preferably, immunohistochemistry and histochemistry is performed onascites and pleural effusion of patients to analyze structuralcharacteristics of the neoplasia. Such methods are known to one skilledin the art and encompassed within the invention. The markers that may bemonitored include for example cytokeratin (to identify ovarianneoplastic and mesothelial cells from inflammatory and mesenchymalcells); calretinin (to separate mesothelial from Her2neu positiveneoplastic cells); and CD45 (to separate inflammatory cells from therest of the cell population in the samples). Additional markers that maybe followed include CD3 (T cells), CD20 (B cells), CD56 (NK cells), andCD14 (monocytes). It will be appreciated by one skilled in the art thatthe immunohistochemistry and histochemistry methods described supra, areanalogously applied to any tumor cell for use in the methods of theinvention. After s.c. inoculation of tumor cells, mice are followed forclinical and anatomical changes. As needed, mice may be necropsied tocorrelate total tumor burden with specific organ localization.

In a specific embodiment, tumors are established using carcinoma celllines such as IGROV-1, OVCAR-8, SK-B, and OVCAR-3 cells and humanovarian carcinoma ascites and pleural effusion from breast cancerpatients. The ascites preferably contain both the effectors and thetumor targets for the antibodies being tested. Human monocytes will betransferred as effectors.

The in vivo activity of the antibodies of the invention may also betested in an animal model, e.g., Balb/c nude mice, injected with cellsexpressing FcγRIIB, including but not limited to SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al., 1976, Cancer Res.33-41); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Rajicells with ATCC accession number CCL-86 (see, e.g., Epstein et al.,1965, J. Natl. Cancer Inst. 34: 231-240), Daudi cells with ATCCaccession number CCL-213 (see, e.g., Klein et al., 1968, Cancer Res. 28:1300-10); ovarian carcinoma cell lines, e.g., OVCAR-3 with ATCCaccession number HTB-161 (see, e.g., Hamilton, Young et al., 1983),SK-OV-3, PA-1, CAOV3, OV-90, and IGROV-1 (available from the NCIrepository Benard et al., 1985, Cancer Research, 45:4970-9; which isincorporated herein by reference in its entirety.

An exemplary assay for measuring the in vivo activity of the antibodiesof the invention may comprise the following: Balb/c Nude female mice(Taconic, MD) are injected at day 0 with cells expressing FcγRIIB suchas 5×10⁶ Daudi cells for example by the subcutaneous route. Mice (e.g.,5 mice per group) also receive i.p. injection of PBS (negative control),ch 4.4.20 (anti-FITC antibody) as a negative control, and as a positivecontrol another therapeutic cancer antibody such as those disclosedherein, e.g., Rituxan, (e.g., at 10 μg/g) or 10 μg/g ch8B5.3.4 once aweek starting at day 0. Mice are observed, e.g., twice a week followinginjection, and tumor size (length and width) is determined using forexample a caliper. Tumor weight in mg is estimated using the formula:(length×width)/2.

Preferably, the antibodies of the invention have an enhanced efficacy indecreasing tumor relative to a cancer therapeutic antibody whenadministered at the same dose, e.g., 10 μg/g, over a time period of atleast 14 days, at least 21 days, at least 28 days, or at least 35 days.In most preferred embodiments, the antibodies of the invention reducetumor size by at least 10 fold, at least 100 fold, at least 1000 foldrelative to administration of a cancer therapeutic antibody at the samedose. In yet another preferred embodiment, the antibodies of theinvention completely abolish the tumor.

5.3.1 Polynucleotides Encoding an Antibody

The present invention also includes polynucleotides that encode theantibodies of the invention (e.g., mouse monoclonal antibody producedfrom hybridoma clone 8B5.3.4, 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2having ATCC accession numbers PTA-7610, PTA-4591, PTA-4592, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively), or othermonoclonal antibodies produced by immunization methods of the invention,and humanized versions thereof, and methods for producing same.

The present invention encompass the polynucleotide encoding the variableheavy chain of the 8B5.3.4 antibody, with ATCC accession numberPTA-7610, as disclosed in SEQ ID NO: 2. The present invention alsoencompasses the polynucleotide encoding the variable light chain of the8B5.3.4 antibody with ATCC accession number PTA-7610, as disclosed inSEQ ID NO: 1.

The methods of the invention also encompass polynucleotides thathybridize under various stringency, e.g., high stringency, intermediateor lower stringency conditions, to polynucleotides that encode anantibody of the invention. The hybridization can be performed undervarious conditions of stringency. By way of example and not limitation,procedures using conditions of low stringency are as follows (see alsoShilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792).Filters containing DNA are pretreated for 6 h at 40° C. in a solutioncontaining 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA,0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA.Hybridizations are carried out in the same solution with the followingmodifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe isused. Filters are incubated in hybridization mixture for 18-20 h at 40°C., and then washed for 1.5 h at 55° C. in a solution containing 2×SSC,25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations). By way ofexample and not limitation, procedures using conditions of highstringency are as follows. Prehybridization of filters containing DNA iscarried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC,50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA,and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48h at 65° C. in prehybridization mixture containing 100 μg/ml denaturedsalmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing offilters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.01%PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSCat 50° C. for 45 min before autoradiography. Other conditions of highstringency which may be used are well known in the art. Selection ofappropriate conditions for such stringencies is well known in the art(see e.g., Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; see also, Ausubel et al., eds., in the Current Protocols inMolecular Biology series of laboratory technique manuals, © 1987-1997,Current Protocols, © 1994-1997 John Wiley and Sons, Inc.; seeespecially, Dyson, 1991, “Immobilization of nucleic acids andhybridization analysis,” In: Essential Molecular Biology: A PracticalApproach, Vol. 2, T. A. Brown, ed., pp. 111-156, IRL Press at OxfordUniversity Press, Oxford, UK).

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art.

A polynucleotide encoding an antibody may be generated from nucleic acidfrom a suitable source (e.g., a cDNA library generated from, or nucleicacid, preferably poly A+RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody of the invention, e.g., 8B5.3.4) by hybridization with Igspecific probes and/or PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., 1990,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998,Current Protocols in Molecular Biology, John Wiley & Sons, NY, which areboth incorporated by reference herein in their entireties), to generateantibodies having a different amino acid sequence, for example to createamino acid substitutions, deletions, and/or insertions.

In a specific embodiment, one or more of the CDRs are inserted withinframework regions using routine recombinant DNA techniques. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal., 1998, J. Mol. Biol. 278: 457-479 for a listing of human frameworkregions). Preferably, the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody that specificallybinds to FcγRIIB with greater affinity than said antibody binds FcγRIIA.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibodies of the invention toFcγRIIB

In another embodiment, human libraries or any other libraries availablein the art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the antibodies of the invention.

5.3.2 Recombinant Expression of Antibodies

Once a nucleic acid sequence encoding an antibody of the invention hasbeen obtained, the vector for the production of the antibody may beproduced by recombinant DNA technology using techniques well known inthe art. Methods which are well known to those skilled in the art can beused to construct expression vectors containing the antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.(See, for example, the techniques described in Sambrook et al., 1990,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998,Current Protocols in Molecular Biology, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of an antibodycan be transferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation) and the transfected cells are then cultured byconventional techniques to produce the antibody of the invention. Inspecific embodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

The host cells used to express the recombinant antibodies of theinvention may be either bacterial cells such as Escherichia coli, or,preferably, eukaryotic cells, especially for the expression of wholerecombinant immunoglobulin molecule. In particular, mammalian cells suchas Chinese hamster ovary cells (CHO), in conjunction with a vector suchas the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Foecking et al., 1998, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

A variety of host-expression vector systems may be utilized to expressthe antibodies of the invention. Such host-expression systems representvehicles by which the coding sequences of the antibodies may be producedand subsequently purified, but also represent cells which may, whentransformed or transfected with the appropriate nucleotide codingsequences, express the antibodies of the invention in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing immunoglobulincoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing immunoglobulin codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the immunoglobulincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing immunoglobulin coding sequences;or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells,lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (ratretinal cells developed by Crucell)) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibodycoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to a matrix glutathione-agarose beads followed byelution in the presence of free gluta-thione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., thepolyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (e.g., seeLogan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibodies of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, 3:87-95; Tolstoshev,1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; May, 1993, TIB TECH 11(5):155-215). Methods commonly knownin the art of recombinant DNA technology which can be used are describedin Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology,John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY; and in Chapters 12 and 13,Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, JohnWiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1;and hygro, which confers resistance to hygromycin (Santerre et al.,1984, Gene 30:147).

The expression levels of an antibody of the invention can be increasedby vector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing an antibodyis amplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of theantibody, production of the antibody will also increase (Crouse et al.,1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once the antibody of the invention has been recombinantly expressed, itmay be purified by any method known in the art for purification of anantibody, for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins.

5.4 Prophylactic and Therapeutic Methods

The present invention encompasses antibody-based therapies which involveadministering one or more of the antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, forpreventing, treating, or ameliorating symptoms associated with adisease, disorder, or infection, associated with aberrant levels oractivity of FcγRIIB and/or treatable by altering immune functionassociated with FcγRIIB activity or enhancing cytotoxic activity of asecond therapeutic antibody or enhancing efficacy of a vaccinecomposition. In some embodiments, therapy by administration of one ormore antibodies of the invention is combine with administration of oneor more therapies such as, but not limited to, chemotherapies, radiationtherapies, hormonal therapies, and/or biologicaltherapies/immunotherapies.

Antibodies may be provided in pharmaceutically acceptable compositionsas known in the art or as described herein. As detailed below, theantibodies of the invention can be used in methods of treating cancer(particularly to enhance passive immunotherapy or efficacy of a cancervaccine), autoimmune disease, inflammatory disorders or allergies (e.g.,to enhance efficacy of a vaccine for treatment of allergy).

FcγRIIB (CD32B) has been found to be expressed in the following tissuetypes: adipose, b-cell, bone, brain, cartilage, colon, endocrine, eye,fetus, gastrointestinal tract, genitourinary, germ cell, head and neck,kidney, lung, lymph node, lymphoreticular, mammary gland, muscle,nervous, ovary, pancreas, pancreatic islet, pituitary gland, placenta,retina, skin, soft tissue, synovium, and uterus (data collected from theCancer Genome Anatomy Project of the National Cancer Institute). Thus,the antibodies of the invention can be used to agonize or antagonize theactivity of FcγRIIB in any of these tissues. For example, FcγRIIB isexpressed in the placenta and may play a role in transport of IgG to thefetus and also in scavenging immune complexes (Lyden et al., 2001, J.Immunol. 166:3882-3889). In certain embodiments of the invention, ananti-FcγRIIB antibody can used as an abortifacient.

The present inventors have found that neutrophils surprisingly do notexpress significant levels of FCγRIIB Accordingly, the inventionprovides methods and pharmaceutical compositions for use in thesemethods, comprising an amount of CD32-specific antibody that binds toand has activity on tumor cells or non-neutrophil cell types, such asmacrophages, but does not detectably bind or have detectable activity onneutrophils. In certain embodiments, the antibodies of the invention canbe used to deplete CD32B⁺ cells, such as macrophages or CD32B-expressingtumor cells.

Antibodies of the present invention that function as a prophylactic andor therapeutic agent of a disease, disorder, or infection can beadministered to an animal, preferably a mammal, and most preferably ahuman, to treat, prevent or ameliorate one or more symptoms associatedwith the disease, disorder, or infection. Antibodies of the inventioncan be administered in combination with one or more other prophylacticand/or therapeutic agents useful in the treatment, prevention ormanagement of a disease, disorder, or infection associated with aberrantlevels or activity of FcγRIIB and/or treatable by altering immunefunction associated with FcγRIIB activity. In certain embodiments, oneor more antibodies of the invention are administered to a mammal,preferably a human, concurrently with one or more other therapeuticagents useful for the treatment of cancer. The term “concurrently” isnot limited to the administration of prophylactic or therapeutic agentsat exactly the same time, but rather it is meant that antibodies of theinvention and the other agent are administered to a subject in asequence and within a time interval such that the antibodies of theinvention can act together with the other agent to provide an increasedbenefit than if they were administered otherwise. For example, eachprophylactic or therapeutic agent may be administered at the same timeor sequentially in any order at different points in time; however, ifnot administered at the same time, they should be administeredsufficiently close in time so as to provide the desired therapeutic orprophylactic effect. Each therapeutic agent can be administeredseparately, in any appropriate form and by any suitable route.

In various embodiments, the prophylactic or therapeutic agents areadministered less than 1 hour apart, at about 1 hour apart, at about 1hour to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, no more than 24 hours apart or no more than 48 hours apart.In preferred embodiments, two or more components are administered withinthe same patient visit.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference (56^(th) ed., 2002).

The antibodies of this invention may also be advantageously utilized incombination with other monoclonal or chimeric antibodies, Fc fusionproteins, or with lymphokines, cytokines or hematopoietic growth factors(such as, e.g., IL-2, IL-3, IL-4, IL-7, IL-10 and TGF-β), which enhanceFcγRIIB, for example, serve to increase the number or activity ofeffector cells which interact with the antibodies and, increase immuneresponse. In certain embodiments, a cytokine is conjugated to ananti-FcγRIIB antibody.

The antibodies of this invention may also be advantageously utilized incombination with one or more drugs used to treat a disease, disorder, orinfection such as, for example anti-cancer agents, anti-inflammatoryagents or anti-viral agents, e.g., as detailed in sections 5.4.6 and5.4.5 below.

5.4.1 Cancers

Antibodies of the invention can be used alone or in combination withother therapeutic antibodies known in the art to prevent, inhibit orreduce the growth of primary tumors or metastasis of cancerous cells. Inone embodiment, antibodies of the invention can be used in combinationwith antibodies used in cancer immunotherapy. The invention encompassesthe use of the antibodies of the invention in combination with anothertherapeutic antibody to enhance the efficacy of such immunotherapy byincreasing the potency of the therapeutic antibody's effector function,e.g., ADCC, CDC, phagocytosis, opsonization, etc. Although not intendingto be bound by a particular mechanism of action antibodies of theinvention block FcγRIIB, preferably on monocytes and macrophages andthus enhance the therapeutic benefits a clinical efficacy of tumorspecific antibodies by, for example, enhancing clearance of the tumorsmediated by activating FcγRs. Accordingly, the invention providesmethods of preventing or treating cancer characterized by a cancerantigen, when administered in combination with another antibody thatspecifically binds a cancer antigen and is cytotoxic. The antibodies ofthe invention are useful for prevention or treatment of cancer,particularly in potentiating the cytotoxic activity of cancerantigen-specific therapeutic antibodies with cytotoxic activity toenhance tumor cell killing by the antibodies of the invention and/orenhancing for example, ADCC activity or CDC activity of the therapeuticantibodies. In certain embodiments of the invention, antibodies of theinvention are administered with Fc fusion proteins. In a specificembodiment, an antibody of the invention, when administered alone or incombination with a cytotoxic therapeutic antibody, inhibits or reducesthe growth of primary tumor or metastasis of cancerous cells by at least99%, at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 60%, at least 50%, at least 45%, at least40%, at least 45%, at least 35%, at least 30%, at least 25%, at least20%, or at least 10% relative to the growth of primary tumor ormetastasis in absence of said antibody of the invention. In a preferredembodiment, antibodies of the invention in combination with a cytotoxictherapeutic antibody inhibit or reduce the growth of primary tumor ormetastasis of cancer by at least 99%, at least 95%, at least 90%, atleast 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to thegrowth or metastasis in absence of said antibodies.

The transition from a normal to a malignant state is a multistep processinvolving genetic and epigenetic changes. In fact, numerous alterationsoccur in the cellular regulatory circuits that facilitate thisprogression which enables tumor cells to evade the commitment toterminal differentiation and quiescence that normally regulate tissuehomeostasis. Certain genes have been implicated in invasiveness andmetastatic potential of cancer cells such as CSF-1 (colony stimulatingfactor 1 or macrophage colony stimulating factor). Although notintending to be bound by a particular mechanism of action, CSF-1 maymediate tumor progression and metastasis by recruiting macrophages tothe tumor site where they promote progression of tumor. It is believedthat macrophages have a trophic role in mediating tumor progression andmetastasis perhaps by the secretion of angiogenic factors, e.g.,thymidine phosphorylase, vascular endothelial-derived growth factor;secretion of growth factors such as epidermal growth factor that couldact as a paracrine factor on tumor cells, and thus promoting tumor cellmigration and invasion into blood vessels. (See, e.g., Lin et al., 2001,J. Exp. Med. 193(6): 727-739; Lin et al., 2002, Journal of Mammary GlandBiology and Neoplasam 7(2): 147-162; Scholl et al., 1993, MolecularCarcinogenesis, 7: 207-11; Clynes et al., 2000, Nature Medicine, 6(4):443-446; Fidler et al., 1985, Cancer Research, 45: 4714-26).

The invention encompasses using the antibodies of the invention to blockmacrophage mediated tumor cell progression and metastasis. Theantibodies of the invention are particularly useful in the treatment ofsolid tumors, where macrophage infiltration occurs. The antagonisticantibodies of the invention are particularly useful for controlling,e.g., reducing or eliminating, tumor cell metastasis, by reducing oreliminating the population of macrophages that are localized at thetumor site. In some embodiments, the antibodies of the invention areused alone to control tumor cell metastasis. Although not intending tobe bound by a particular mechanism of action the antagonistic antibodiesof the invention, when administered alone bind the inhibitory FcγRIIB onmacrophages and effectively reduce the population of macrophages andthus restrict tumor cell progression. The antagonistic antibodies of theinvention reduce, or preferably eliminate macrophages that are localizedat the tumor site, since FcγRIIB is preferentially expressed onactivated monocytes and macrophages including tumor-infiltratingmacrophages. In some embodiments, the antibodies of the invention areused in the treatment of cancers that are characterized by theoverexpression of CSF-1, including but not limited to breast, uterine,and ovarian cancers.

The invention further encompasses antibodies that effectively deplete oreliminate immune cells other than macrophages that express FcγRIIB,e.g., dendritic cells and B-cells. Effective depletion or elimination ofimmune cells using the antibodies of the invention may range from areduction in population of the immune cells by 50%, 60%, 70%, 80%,preferably 90%, and most preferably 99%. Thus, the antibodies of theinvention have enhanced therapeutic efficacy either alone or incombination with a second antibody, e.g., a therapeutic antibody such asanti-tumor antibodies, anti-viral antibodies, and anti-microbialantibodies. In some embodiments, the therapeutic antibodies havespecificity for a cancer cell or an inflammatory cell. In otherembodiments, the second antibody binds a normal cell. Although notintending to be bound by a particular mechanism of action, when theantibodies of the invention are used alone to deplete FcγRIIB-expressingimmune cells, the population of cells is redistributed so thateffectively the cells that are remaining have the activating Fcreceptors and thus the suppression by FcγRIIB is alleviated. When usedin combination with a second antibody, e.g., a therapeutic antibody theefficacy of the second antibody is enhanced by increasing theFc-mediated effector function of the antibody.

Cancers and related disorders that can be treated or prevented bymethods and compositions of the present invention include, but are notlimited to, the following: Leukemias including, but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome, chronicleukemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytom and adrenocortical carcinoma; thyroid cancer such asbut not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including butnot limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including but not limited tohepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding but not limited to, adenocarcinoma; cholangiocarcinomasincluding but not limited to, pappillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including but not limitedto, squamous cell cancer, and verrucous; skin cancers including but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including but notlimited to, renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers including but not limited to, transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosafcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include but not belimited to follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

Cancers associated with the cancer antigens may be treated or preventedby administration of the antibodies of the invention in combination withan antibody that binds the cancer antigen and is cytotoxic. In oneparticular embodiment, the antibodies of the invention enhance theantibody mediated cytotoxic effect of the antibody directed at theparticular cancer antigen. For example, but not by way of limitation,cancers associated with the following cancer antigen may be treated orprevented by the methods and compositions of the invention. KS 1/4pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:32-37;Bumal, 1988, Hybridoma 7(4):407-415), ovarian carcinoma antigen (CA125)(Yu et al., 1991, Cancer Res. 51(2):48-475), prostatic acid phosphate(Tailor et al., 1990, Nucl. Acids Res. 18(1):4928), prostate specificantigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm.10(2):903-910; Israeli et al., 1993, Cancer Res. 53:227-230),melanoma-associated antigen p97 (Estin et al., 1989, J. Natl. CancerInstit. 81(6):445-44), melanoma antigen gp75 (Vijayasardahl et al.,1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanomaantigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-3; Mittelman etal., 1990, J. Clin. Invest. 86:2136-2144)), prostate specific membraneantigen, carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am.Soc. Clin. Oncol. 13:294), polymorphic epithelial mucin antigen, humanmilk fat globule antigen, Colorectal tumor-associated antigens such as:CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), CO17-1A(Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlynet al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA, Burkitt'slymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336),human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445),CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430), melanoma specificantigens such as ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151,3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol.Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1994, J.Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, CancerRes. 53:5244-5250), tumor-specific transplantation type of cell-surfaceantigen (TSTA) such as virally-induced tumor antigens includingT-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses,oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumoroncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185^(HER2)),polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio.Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhardet al., 1989, Science 245:301-304), differentiation antigen (Feizi,1985, Nature 314:53-57) such as I antigen found in fetal erthrocytes andprimary endoderm, I(Ma) found in gastric adencarcinomas, M18 and M39found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9,Myl, VIM-D5, and D156-22 found in colorectal cancer, TRA-1-85 (bloodgroup H), C14 found in colonic adenocarcinoma, F3 found in lungadenocarcinoma, AH6 found in gastric cancer, Y hapten, Le^(y) found inembryonal carcinoma cells, TL5 (blood group A), EGF receptor found inA431 cells, E₁ series (blood group B) found in pancreatic cancer, FC10.2found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514(blood group Lea) found in adenocarcinoma, NS-10 found inadenocarcinomas, CO-43 (blood group Le^(b)), G49, EGF receptor, (bloodgroup ALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 found incolon cancer, gastric cancer mucins, T5A7 found in myeloid cells, R24found in melanoma, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, M1:22:25:8found in embryonal carcinoma cells and SSEA-3, SSEA-4 found in 4-8-cellstage embryos. In another embodiment, the antigen is a T cell receptorderived peptide from a cutaneous T cell lymphoma (see Edelson, 1998, TheCancer Journal 4:62).

The antibodies of the invention can be used in combination with anytherapeutic cancer antibodies known in the art to enhance the efficacyof treatment. For example, the antibodies of the invention can be usedwith any of the antibodies in Table 7, that have demonstratedtherapeutic utility in cancer treatment. The antibodies of the inventionenhance the efficacy of treatment of the therapeutic cancer antibodiesby enhancing at least one antibody-mediated effector function of saidtherapeutic cancer antibodies. In one particular embodiment, theantibodies enhance the efficacy of treatment by enhancing the complementdependent cascade of said therapeutic cancer antibodies. In anotherembodiment of the invention, the antibodies of the invention enhance theefficacy of treatment by enhancing the phagocytosis and opsonization ofthe targeted tumor cells. In another embodiment of the invention, theantibodies of the invention enhance the efficacy of treatment byenhancing antibody-dependent cell-mediated cytotoxicity (“ADCC”) indestruction of the targeted tumor cells.

Antibodies of the invention can also be used in combination withcytosine-guanine dinucleotides (“CpG”)-based products that have beendeveloped (Coley Pharmaceuticals) or are currently being developed asactivators of innate and acquired immune responses. For example, theinvention encompasses the use of CpG 7909, CpG 8916, CpG 8954 (ColeyPharmaceuticals) in the methods and compositions of the invention forthe treatment and/or prevention of cancer (See also Warren et al., 2002,Semin Oncol., 29(1 Suppl 2):93-7; Warren et al., 2000, Clin Lymphoma,1(1):57-61, which are incorporated herein by reference).

Antibodies of the invention can be used in combination with atherapeutic antibody that does not mediate its therapeutic effectthrough cell killing to potentiate the antibody's therapeutic activity.In a specific embodiment, the invention encompasses use of theantibodies of the invention in combination with a therapeutic apoptosisinducing antibody with agonistic activity, e.g., an anti-Fas antibody.Anti-Fas antibodies are known in the art and include for example, Jo2(Ogasawara et al., 1993, Nature 364: 806) and HFE7 (Ichikawa et al.,2000, Int. Immunol. 12: 555). Although not intending to be bound by aparticular mechanisms of action, FcγRIIB has been implicated inpromoting anti-Fas mediated apoptosis, see, e.g., Xu et al., 2003,Journal of Immunology, 171: 562-568. In fact the extracellular domain ofFcγRIIB may serve as a cross-linking agent for Fas receptors, leading toa functional complex and promoting Fas dependent apoptosis. In someembodiments, the antibodies of the invention block the interaction ofanti-Fas antibodies and FcγRIIB, leading to a reduction in Fas-mediatedapoptotic activity. Antibodies of the invention that result in areduction in Fas-mediated apoptotic activity are particularly useful incombination with anti-Fas antibodies that have undesirable side effects,e.g., hepatotoxicity. In other embodiments, the antibodies of theinvention enhance the interaction of anti-Fas antibodies and FcγRIIB,leading to an enhancement of Fas-mediated apoptotic activity.Combination of the antibodies of the invention with therapeuticapoptosis inducing antibodies with agonistic activity have an enhancedtherapeutic efficacy.

Therapeutic apoptosis inducing antibodies used in the methods of theinvention may be specific for any death receptor known in the art forthe modulation of apoptotic pathway, e.g., TNFR receptor family.

The invention provides a method of treating diseases with impairedapoptotic mediated signaling, e.g., cancer, autoimmune disease. In aspecific embodiment, the invention encompasses a method of treating adisease with deficient Fas-mediated apoptosis, said method comprisingadministering an antibody of the invention in combination with ananti-Fas antibody.

In some embodiments, the agonistic antibodies of the invention areparticularly useful for the treatment of tumors of non-hematopoieticorigin, including tumors of melanoma cells. Although not intending to bebound by a particular mechanism of action, the efficacy of the agonisticantibodies of the invention is due, in part, to activation of FcγRIIBinhibitory pathway, as tumors of non-hematopoietic origin, includingtumors of melanoma cells express FcγRIIB Recent experiments have in factshown that expression of FcγRIIB in melanoma cells modulates tumorgrowth by direct interaction with anti-tumor antibodies (e.g., bybinding the Fc region of the anti-tumor antibodies) in anintracytoplasmic-dependent manner (Cassard et al., 2002, Journal ofClinical Investigation, 110(10): 1549-1557).

In some embodiments, the invention encompasses use of the antibodies ofthe invention in combination with therapeutic antibodies thatimmunospecifically bind to tumor antigens that are not expressed on thetumor cells themselves, but rather on the surrounding reactive and tumorsupporting, non-malignant cells comprising the tumor stroma. The tumorstroma comprises endothelial cells forming new blood vessels and stromalfibroblasts surrounding the tumor vasculature. In a specific embodiment,an antibody of the invention is used in combination with an antibodythat immunospecifically binds a tumor antigen on an endothelial cell. Ina preferred embodiment, an antibody of the invention is used incombination with an antibody that immunospecifically binds a tumorantigen on a fibroblast cell, e.g., fibroblast activation protein (FAP).FAP is a 95 KDa homodimeric type II glycoprotein which is highlyexpressed in stromal fibroblasts of many solid tumors, including, butnot limited to lung, breast, and colorectal carcinomas. (See, e.g.,Scanlan et al., 1994; Proc. Natl. Acad. USA, 91: 5657-61; Park et al.,1999, J. Biol. Chem., 274: 36505-12; Rettig et al., 1988, Proc. Natl.Acad. Sci. USA 85: 3110-3114; Garin-Chesea et al., 1990, Proc. Natl.Acad. Sci. USA 87: 7235-7239). Antibodies that immunospecifically bindFAP are known in the art and encompassed within the invention, see,e.g., Wuest et al., 2001, Journal of Biotechnology, 159-168; Mersmann etal., 2001, Int. J. Cancer, 92: 240-248; U.S. Pat. No. 6,455,677; all ofwhich are incorporated herein in by reference in their entireties.

Recently IgE's have been implicated as mediators of tumor growth and infact IgE-targeted immediate hypersensitivity and allergic inflammationreactions have been proposed as possible natural mechanisms involved inanti-tumor responses (For a review see, e.g., Mills et al., 1992, Am.Journal of Epidemiol. 122: 66-74; Eriksson et al., 1995, Allergy 50:718-722). In fact a recent study has shown loading tumor cells with IgEsreduces tumor growth, leading in some instances to tumor rejection.According to the study, IgE loaded tumor cells not only possess atherapeutic potential but also confer long term antitumor immunity,including activation of innate immunity effector mechanism and T-cellmediated adaptive immune response, see Reali et al., 2001, Cancer Res.61: 5516-22; which is incorporated herein by reference in its entirety.The antagonistic antibodies of the invention may be used in thetreatment and/or prevention of cancer in combination with administrationof IgEs in order to enhance the efficacy of IgE-mediated cancer therapy.Although not intending to be bound by a particular mechanism of actionthe antibodies of the invention enhance the therapeutic efficacy of IgEtreatment of tumors, by blocking the inhibitory pathway. Theantagonistic antibodies of the invention may enhance the therapeuticefficacy of IgE-mediated cancer therapy by (i) enhancing the delay intumor growth; (ii) enhancing the decrease in the rate of tumorprogression; (iii) enhancing tumor rejection; or (iv) enhancingprotective immune relative to treatment of cancer with IgE alone.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in theliterature, see, e.g., Physician's Desk Reference (56^(th) ed., 2002,which is incorporated herein by reference).

5.4.1.1 B Cell Malignancies

The present invention encompasses therapies which involve administeringan anti-FcγRIIB antibody, to an animal, preferably a mammal, and mostpreferably a human, to prevent, treat, manage or ameliorate a B-cellmalignancy, or one or more symptoms thereof. These therapies are anenhancement over current therapies. In certain cases, patients who arerefractory to current therapies can be treated with the methods of theinvention. In some embodiments, therapy by administration of one or moreantibodies of the invention is combined with administration of one ormore therapies such as, but not limited to, chemotherapies, radiationtherapies, hormonal therapies, and/or biologicaltherapies/immunotherapies.

The present invention encompasses treatment protocols that providebetter prophylactic and therapeutic profiles than current single agenttherapies or combination therapies for a B-cell malignancy, or one ormore symptoms thereof. The invention provides FcγRIIB antibody basedtherapies for the prevention, treatment, management, or amelioration ofa B-cell malignancy, or one or more symptoms thereof. In particular, theinvention provides prophylactic and therapeutic protocols for theprevention, treatment, management, or amelioration of a B-cellmalignancy, or one or more symptoms thereof, comprising theadministration of a FcγRIIB-specific antibody, an analog, derivative oran antigen-fragment thereof to a subject in need thereof.

The agonistic antibodies of the invention are useful for treating orpreventing any B cell malignancies, particularly non-Hodgkin's lymphomaand chronic lymphocytic leukemia. Other B-cell malignancies includesmall lymphocytic lymphoma, Burkitt's lymphoma, mantle cell lymphomasdiffuse small cleaved cell lymphomas, most follicular lymphomas and somediffuse large B cell lymphomas (DLBCL). FcγRIIB, is a target forderegulation by chromosomal translocation in malignant lymphoma,particularly in B-cell non-Hodgkin's lymphoma (See Callanan M. B. etal., 2000 Proc. Natl. Acad. Sci. U.S.A., 97(1):309-314). Thus, theantibodies of the invention are useful for treating or preventing anychronic lymphocytic leukemia of the B cell lineage. Chronic lymphocyticleukemia of the B cell lineage are reviewed by Freedman (See review byFreedman, 1990, Hemtaol. Oncol. Clin. North Am. 4:405). Although notintending to be bound by any mechanism of action, the agonisticantibodies of the invention inhibit or prevent B cell malignanciesinhibiting B cell proliferation and/or activation. The invention alsoencompasses the use of the agonistic antibodies of the invention incombination with other therapies known (e.g., chemotherapy andradiotherapy) in the art for the prevention and/or treatment of B cellmalignancies. The invention also encompasses the use of the agonisticantibodies of the invention in combination with other antibodies knownin the art for the treatment and or prevention of B-cell malignancies.For example, the agonistic antibodies of the invention can be used incombination with the anti-C22 or anti-CD19 antibodies disclosed byGoldenberg et al. (U.S. Pat. No. 6,306,393), anti-CD20 antibodies,anti-CD33 antibodies, or anti-CD52 antibodies.

Antibodies of the invention can also be used in combination with forexample but not by way of limitation, Oncoscint (target: CEA), Verluma(target: GP40), Prostascint® (target: PSMA), CEA-SCAN™ (target: CEA),Rituxan® (target: CD20), Herceptin® (target: HER-2), Campath® (target:CD52), Mylotarge™ (target: CD33), LymphoCide (CD22), Lymphocide Y-90™(CD22) and Zevalin® (target: CD20).

5.4.2 Autoimmune Disease and Inflammatory Diseases

The agonistic antibodies of the invention may be used to treat orprevent autoimmune diseases or inflammatory diseases. The presentinvention provides methods of preventing, treating, or managing one ormore symptoms associated with an autoimmune or inflammatory disorder ina subject, comprising administering to said subject a therapeuticallyeffective amount of the antibodies or fragments thereof of theinvention. The invention also provides methods for preventing, treating,or managing one or more symptoms associated with an inflammatorydisorder in a subject further comprising, administering to said subjecta therapeutically effective amount of one or more anti-inflammatoryagents. The invention also provides methods for preventing, treating, ormanaging one or more symptoms associated with an autoimmune diseasefurther comprising, administering to said subject a therapeuticallyeffective amount of one or more immunomodulatory agents. Section 5.4.5provides non-limiting examples of anti-inflammatory agents andimmunomodulatory agents.

The antibodies of the invention can also be used in combination with anyof the antibodies known in the art for the treatment and/or preventionof autoimmune disease or inflammatory disease. A non-limiting example ofthe antibodies or Fc fusion proteins that are used for the treatment orprevention of inflammatory disorders is presented in Table 6A, and anon-limiting example of the antibodies or Fc fusion proteins that areused for the treatment or prevention of autoimmune disorder is presentedin Table 6B. The antibodies of the invention can for example, enhancethe efficacy of treatment of the therapeutic antibodies or Fc fusionproteins presented in Tables 6A and 6B. For example, but not by way oflimitation, the antibodies of the invention can enhance the immuneresponse in the subject being treated with any of the antibodies or Fcfusion proteins in Tables 6A or 6B.

Antibodies of the invention can also be used in combination with forexample but not by way of limitation, Orthoclone OKT3®, ReoPro,Zenapax®, Simulec, Rituximab, Synagis®, and Remicade®.

Antibodies of the invention can also be used in combination withcytosine-guanine dinucleotides (“CpG”)-based products that have beendeveloped (Coley Pharmaceuticals) or are currently being developed asactivators of innate and acquired immune responses. For example, theinvention encompasses the use of CpG 7909, CpG 8916, CpG 8954 (ColeyPharmaceuticals) in the methods and compositions of the invention forthe treatment and/or prevention of autoimmune or inflammatory disorders(Weeratna et al., 2001, FEMS Immunol Med Microbiol., 32(1):65-71, whichis incorporated herein by reference).

Examples of autoimmune disorders that may be treated by administeringthe antibodies of the present invention include, but are not limited to,alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Meniere's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteristis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. As described herein inSection 3.1, some autoimmune disorders are associated with aninflammatory condition. Thus, there is overlap between what isconsidered an autoimmune disorder and an inflammatory disorder.Therefore, some autoimmune disorders may also be characterized asinflammatory disorders. Examples of inflammatory disorders which can beprevented, treated or managed in accordance with the methods of theinvention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections.

In certain embodiments of the invention, the antibodies of the inventionmay be used to treat an autoimmune disease that is more prevalent in onesex. For example, the prevalence of Graves' disease in women has beenassociated with expression of FcγRIIB2 (see Estienne et al., 2002, FASEBJ. 16:1087-1092).

Antibodies of the invention can also be used to reduce the inflammationexperienced by animals, particularly mammals, with inflammatorydisorders. In a specific embodiment, an antibody reduces theinflammation in an animal by at least 99%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to theinflammation in an animal in the not administered said antibody. Inanother embodiment, a combination of antibodies reduce the inflammationin an animal by at least 99%, at least 95%, at least 90%, at least 85%,at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10% relative to the inflammation inan animal in not administered said antibodies.

TABLE 6A Antibodies for Inflammatory Diseases and Autoimmune Diseasesthat can be used in combination with the antibodies of the invention.Antibody Target Name Antigen Product Type Isotype Sponsors Indication5G1.1 Complement Humanised IgG Alexion Rheumatoid (C5) Pharm IncArthritis 5G1.1 Complement Humanised IgG Alexion SLE (C5) Pharm Inc5G1.1 Complement Humanised IgG Alexion Nephritis (C5) Pharm Inc 5G1.1-SCComplement Humanised ScFv Alexion Cardiopulmano (C5) Pharm Inc Bypass5G1.1-SC Complement Humanised ScFv Alexion Myocardial (C5) Pharm IncInfarction 5G1.1-SC Complement Humanised ScFv Alexion Angioplasty (C5)Pharm Inc ABX-CBL CBL Human Abgenix Inc GvHD ABX-CBL CD147 Murine IgGAbgenix Inc Allograft rejection ABX-IL8 IL-8 Human IgG2 Abgenix IncPsoriasis Antegren VLA-4 Humanised IgG Athena/Elan Multiple SclerosisAnti-CD11a CD11a Humanised IgG1 Genentech Psoriasis Inc/Xoma Anti-CD18CD18 Humanised Fab′2 Genentech Inc Myocardial infarction Anti-LFA1 CD18Murine Fab′2 Pasteur- Allograft rejection Merieux/ Immunotech AntovaCD40L Humanised IgG Biogen Allograft rejection Antova CD40L HumanisedIgG Biogen SLE BTI-322 CD2 Rat IgG Medimmune GvHD, Psoriasis Inc CDP571TNF-alpha Humanised IgG4 Celltech Crohn's CDP571 TNF-alpha HumanisedIgG4 Celltech Rheumatoid Arthritis CDP850 E-selectin Humanised CelltechPsoriasis Corsevin M Fact VII Chimeric Centocor Anticoagulant D2E7TNF-alpha Human CAT/BASF Rheumatoid Arthritis Hu23F2G CD11/18 HumanisedICOS Pharm Multiple Sclerosis Inc Hu23F2G CD11/18 Humanised IgG ICOSPharm Stroke Inc IC14 CD14 ICOS Pharm Toxic shock Inc ICM3 ICAM-3Humanised ICOS Pharm Psoriasis Inc IDEC-114 CD80 Primatised IDECPsoriasis Pharm/Mitsubishi IDEC-131 CD40L Humanised IDEC SLE Pharm/EisaiIDEC-131 CD40L Humanised IDEC Multiple Sclerosis Pharm/Eisai IDEC-151CD4 Primatised IgG1 IDEC Rheumatoid Pharm/Glaxo Arthritis SmithKlineIDEC-152 CD23 Primatised IDEC Pharm Asthma/Allergy Infliximab TNF-alphaChimeric IgG1 Centocor Rheumatoid Arthritis Infliximab TNF-alphaChimeric IgG1 Centocor Crohn's LDP-01 beta2- Humanised IgG MillenniumStroke integrin Inc (LeukoSite Inc.) LDP-01 beta2- Humanised IgGMillennium Allograft rejection integrin Inc (LeukoSite Inc.) LDP-02alpha4beta7 Humanised Millennium Ulcerative Colitis Inc (LeukoSite Inc.)MAK-195F TNF alpha Murine Fab′2 Knoll Pharm, Toxic shock BASF MDX-33CD64 (FcR) Human Medarex/Centeon Autoimmune haematogical disordersMDX-CD4 CD4 Human IgG Medarex/Eisai/ Rheumatoid Genmab ArthritisMEDI-507 CD2 Humanised Medimmune Psoriasis Inc MEDI-507 CD2 HumanisedMedimmune GvHD Inc OKT4A CD4 Humanised IgG Ortho Biotech Allograftrejection OrthoClone ™ CD4 Humanised IgG Ortho Biotech Autoimmune OKT4Adisease Orthoclone ™/ CD3 Murine mIgG2a Ortho Biotech Allograftrejection anti-CD3 OKT3 RepPro ™/ gpIIbIIIa Chimeric Fab Centocor/LillyComplications of Abciximab coronary angioplasty rhuMab- IgE HumanisedIgG1 Genentech/Novartis/ Asthma/Allergy E25 Tanox Biosystems SB-240563IL5 Humanised GlaxoSmithKline Asthma/Allergy SB-240683 IL-4 HumanisedGlaxoSmithKline Asthma/Allergy SCH55700 IL-5 Humanised Celltech/ScheringAsthma/Allergy Simulect CD25 Chimeric IgG1 Novartis Allograft rejectionPharm SMART CD3 Humanised Protein Autoimmune a-CD3 Design Lab diseaseSMART CD3 Humanised Protein Allograft rejection a-CD3 Design Lab SMARTCD3 Humanised IgG Protein Psoriasis a-CD3 Design Lab Zenapax ® CD25Humanised IgG1 Protein Allograft rejection Design Lab/Hoffman- La Roche

TABLE 6B Antibodies and Fc fusion proteins for Autoimmune DisordersAntibody Indication Target Antigen ABX-RB2 antibody to CBL antigen on Tcells, B cells and NK cells fully human antibody from the XenomouseIL1-ra rheumatoid arthritis recombinant anti-inflammatory proteinsTNF-RI chronic inflammatory disease soluble tumor necrosis factor a -rheumatoid arthritis receptor type I blocks TNF action 5c8 (Anti CD-40Phase II trials were halted in October CD-40 ligand antibody) 1999examine “adverse events” IDEC 131 systemic lupus erythyematous anti CD40(SLE) humanized IDEC 151 rheumatoid arthritis primatized; anti-CD4 IDEC152 asthma primatized; anti-CD23 IDEC 114 psoriasis primatized anti-CD80MEDI-507 rheumatoid arthritis; multiple anti-CD2 sclerosis Crohn'sdisease psoriasis LDP-02 (anti-b7 inflammatory bowel disease a4b7integrin receptor on white mAb) Chron's disease blood cells (leukocytes)ulcerative colitis SMART Anti- autoimmune disorders Anti-GammaInterferon Gamma Interferon antibody ™ Verteportin ™ rheumatoidarthritis inhibitor of tumor necrosis factor Thalomid ™ leprosy -approved for market alpha (TNF alpha) (thalidomide) Chron's diseaseSelCIDs (selective rheumatoid arthritis highly specific cytokineinhibitory inhibitors of phosphodiesterase drugs) type 4 enzyme (PDE-4)IMiDs general autoimmune disorders increases levels of cAMP (cyclic(immunomodulatory adenosine monophosphate) drugs) activates proteinkinase A (PKA) blocks transcription factor NK-kB prevents transcriptionof TNF-a gene decreases production of TNF-a structural analogues ofthalidomideinhibit TNF-a MDX-33 ™ blood disorders caused by monoclonalantibody against FcRI autoimmune reactions receptors IdiopathicThrombocytopenia Purpurea (ITP) autoimmune hemolytic anemia MDX-CD4 ™treat rheumatoid arthritis and other monoclonal antibody against CD4autoimmunity receptor molecule VX-497 autoimmune disorders inhibitor ofinosine multiple sclerosis monophosphate dehydrogenase rheumatoidarthritis (enzyme needed to make new inflammatory bowel disease RNA andDNA lupus used in production of nucleotides psoriasis needed forlymphocyte proliferation) VX-740 rheumatoid arthritis inhibitor of ICEinterleukin-1 beta (converting enzyme controls pathways leading toaggressive immune response regulates cytokines) VX-745 specific toinflammation inhibitor of P38MAP kinase involved in chemical signalingof mitogen activated protein kinase immune response onset andprogression of inflammation Enbrel targets TNF (tumor necrosis(etanercept) ® factor) IL-8 fully human MAB against IL-8 (interleukin 8)(blocks IL-8 blocks inflammatory response) 5G1.1 rheumatoid arthritis aC5 complement inhibitor pemphigoid (dangerous skin rash) psoriasis lupusApogen ™ MP4 recombinant antigen selectively destroys disease associatedT-cells induces apoptosis T-cells eliminated by programmed cell death nolonger attack body's own cells specific apogens target specific T- cells

5.4.3 Allergy

The invention provides methods for treating or preventing anIgE-mediated and or FcγRI mediated allergic disorder in a subject inneed thereof, comprising administering to said subject a therapeuticallyeffective amount of the agonistic antibodies or fragments thereof of theinvention. Although not intending to be bound by a particular mechanismof action, antibodies of the invention are useful in inhibitingFcεRI-induced mast cell activation, which contributes to acute and latephase allergic responses (Metcalfe D. et al. 1997, Physiol. Rev.77:1033). Preferably, the agonistic antibodies of the invention haveenhanced therapeutic efficacy and/or reduced side effects in comparisonwith the conventional methods used in the art for the treatment and/orprevention of IgE-mediated allergic disorders. Conventional methods forthe treatment and/or prevention of IgE-mediated allergic disordersinclude, but are not limited to, anti-inflammatory drugs (e.g., oral andinhaled corticosteroids for asthma), antihistamines (e.g., for allergicrhinitis and atopic dermatitis), cysteinyl leukotrienes (e.g., for thetreatment of asthma); anti-IgE antibodies; and specific immunotherapy ordesensitization.

Examples of IgE-mediated allergic responses include, but are not limitedto, asthma, allergic rhinitis, gastrointestinal allergies, eosinophilia,conjunctivitis, atopic dermatitis, urticaria, anaphylaxis, or golmerularnephritis.

The invention encompasses molecules, e.g., immunoglobulins, engineeredto form complexes with FcγRI and human FcγRIIB, i.e., specifically bindFcγRT and human FcγRIIB Preferably, such molecules have therapeuticefficacy in IgE and FcγRT-mediated disorders. Although not intending tobe bound by a particular mechanism of action, the therapeutic efficacyof these engineered molecules is, in part, due to their ability toinhibit mast cell and basophil function.

In a specific embodiment, molecules that specifically bind FcγRI andhuman FcγRIIB are chimeric fusion proteins comprising a binding site forFcγRI and a binding site for FcγRIIB Such molecules may be engineered inaccordance with standard recombinant DNA methodologies known to oneskilled in the art. In a preferred specific embodiment, a chimericfusion protein for use in the methods of the invention comprises anF(ab′) single chain of an anti-FcγRIIB monoclonal antibody of theinvention fused to a region used as a bridge to link the huFcγ□ to theC-terminal region of the F(ab′) single chain of the anti-FcγRIIBmonoclonal antibody. One exemplary chimeric fusion protein for use inthe methods of the invention comprises the following: V_(L)/C_(H)(FcγRIIB)-hinge-V_(H)/C_(H) (FcγRIIB)-LINKER-C_(H)ε2-C_(H)ε3-C_(H)ε4.The linker for the chimeric molecules may be five, ten, preferablyfifteen amino acids in length. The length of the linker may vary toprovide optimal binding of the molecule to both FcγRIIB and FcγRI. In aspecific embodiment, the linker is a 15 amino acid linker, consisting ofthe sequence: (Gly₄Ser)₃ Although not intending to be bound by aparticular mechanism of action, the flexible peptide linker facilitateschain pairing and minimizes possible refolding and it will also allowthe chimeric molecule to reach the two receptors, i.e., FcγRIIB andFcγRI on the cells and cross-link them. Preferably, the chimericmolecule is cloned into a mammalian expression vector, e.g., pCI-neo,with a compatible promoter, e.g., cytomegalovirus promoter. The fusionprotein prepared in accordance with the methods of the invention willcontain the binding site for FcεRI (CHε2CHε3) and for FcγRIIB (VL/CL,-hinge-VH/CH). The nucleic acid encoding the fusion protein prepared inaccordance with the methods of the invention is preferably transfectedinto 293 cells and the secreted protein is purified using common methodsknown in the art.

Binding of the chimeric molecules to both human FcεRI and FcγRIIB may beassessed using common methods known to one skilled in the art fordetermining binding to an FcγR. Preferably, the chimeric molecules ofthe invention have therapeutic efficacy in treating IgE-mediateddisorders, for example, by inhibiting antigen-driven degranulation andinhibition of cell activation. The efficacy of the chimeric molecules ofthe invention in blocking IgE driven FcεRI-mediated mast celldegranulation may be determined in transgenic mice, which have beenengineered to express the human FcεRa and human FcγRIIB, prior to theiruse in humans.

The invention provides the use of bispecific antibodies for thetreatment and/or prevention of IgE-mediated and/or FcγRT-mediatedallergic disorders. A bispecific antibody (BsAb) binds to two differentepitopes usually on distinct antigens. BsAbs have potential clinicalutility and they have been used to target viruses, virally infectedcells and bacterial pathogens as well as to deliver thrombolitic agentsto blood clots (Cao Y., 1998 Bioconj. Chem 9: 635-644; Koelemij et al.,1999, J. Immunother., 22, 514-524; Segal et al., Curr. Opin. Immunol.,11, 558-562). The technology for the production of BsIgG and otherrelated bispecific molecules is available (see, e.g., Carter et al.,2001 J. of Immunol. Methods, 248, 7-15; Segal et al., 2001, J. ofImmunol. Methods, 248, 7-15, which are incorporated herein by referencein their entirety). The instant invention provides bispecific antibodiescontaining one F(ab′) of the anti-FcγRIIB antibody and one F(ab′) of anavailable monoclonal anti-huIgE antibody which aggregates two receptors,FcγRIIB and FcεRI, on the surface of the same cell. Any methodologyknown in the art and disclosed herein may be employed to generatebispecific antibodies for use in the methods of the invention. In aspecific embodiment, the BsAbs will be produced by chemicallycross-linking F(ab′) fragments of an anti-FcγRIIB antibody and ananti-huIgE antibody as described previously, see, e.g., Glennie et al.,1995, Tumor Immunobiology, Oxford University press, Oxford, p. 225;which is incorporated herein by reference in its entirety). The F(ab′)fragments may be produced by limited proteolysis with pepsin and reducedwith mercaptoethanol amine to provide Fab′ fragments with freehinge-region sulfhydryl (SH) groups. The SH group on one of the Fab′(SH) fragments may be alkylated with excess 0-phenylenedimaleimide(0-PDM) to provide a free maleimide group (mal). The two preparationsFab′(mal) and Fab′(SH) may be combined at an appropriate ratio,preferably 1:1 to generate heterodimeric constructs. The BsAbs can bepurified by size exclusion chromatography and characterized by HPLCusing methods known to one skilled in the art.

In particular, the invention encompasses bispecific antibodiescomprising a first heavy chain-light chain pair that binds FcγRIIB withgreater affinity than said heavy chain-light chain pair binds FcγRIIA,and a second heavy chain-light chain pair that binds IgE receptor, withthe provision that said first heavy chain-light chain pair binds FcγRIIBfirst. The bispecific antibodies of the invention can be engineeredusing standard techniques known in the art to ensure that the binding toFcγRIIB precedes the binding to the IgE receptor. It will be understoodto one skilled in the art to engineer the bispecific antibodies, forexample, such that said bispecific antibodies bind FcγRIIB with greateraffinity than said antibodies bind IgE receptor. Additionally, thebispecific antibodies can be engineered by techniques known in the art,such that the hinge size of the antibody can be increased in length, forexample, by adding linkers, to provide the bispecific antibodies withflexibility to bind the IgE receptor and FcγRIIB receptor on the samecell.

The antibodies of the invention can also be used in combination withother therapeutic antibodies or drugs known in the art for the treatmentor prevention of IgE-mediated allergic disorders. For example, theantibodies of the invention can be used in combination with any of thefollowing: azelastine, Astelin™, beclomethasone dipropionate inhaler,Vanceril, beclomethasone dipropionate nasal inhaler/spray, Vancenase®,Beconase budesonide nasal inhaler/spray, Rhinocort® cetirizine, Zyrtec®chlorpheniramine, pseudoephedrine, Deconamine, Sudafed®, cromolyn,Nasalcrom®, Intal®, Opticrom®, desloratadine, Clarinex®, fexofenadineand pseudoephedrine, Allegra-D®, fexofenadine, Allegra® flunisolidenasal spray, Nasalide® fluticasone propionate nasal inhaler/spray,Flonase® fluticasone propionate oral inhaler, Flovent®, hydroxyzine,Vistaril®, Ataraxloratadine®, pseudoephedrine, Claritin-D®, loratadine,Claritin®, prednisolone, Prednisolone, Pediapred® Oral Liquid, Medrol®prednisone, Deltasone®, Liquid Predsalmeterol®, Serevent® triamcinoloneacetonide inhaler, Azmacort® triamcinolone acetonide nasalinhaler/spray, Nasacort®, or NasacortAQ®. Antibodies of the inventioncan be used in combination with cytosine-guanine dinucleotides(“CpG”)-based products that have been developed (Coley Pharmaceuticals)or are currently being developed as activators of innate and acquiredimmune responses. For example, the invention encompasses the use of CpG7909, CpG 8916, CpG 8954 (Coley Pharmaceuticals) in the methods andcompositions of the invention for the treatment and/or prevention ofIgE-mediated allergic disorders (See also Weeratna et al., 2001, FEMSImmunol Med Microbiol., 32(1):65-71, which is incorporated herein byreference).

The invention encompasses the use of the antibodies of the invention incombination with any therapeutic antibodies known in the art for thetreatment of allergy disorders, e.g., Xolair™ (Omalizumab; Genentech);rhuMAB-E25 (BioWorld Today, Nov. 10, 1998, p. 1; Genentech); CGP-51901(humanized anti-IgE antibody), etc.

Additionally, the invention encompasses the use of the antibodies of theinvention in combination with other compositions known in the art forthe treatment of allergy disorders. In particular methods andcompositions disclosed in Carson et al. (U.S. Pat. No. 6,426,336; US2002/0035109 A1; US 2002/0010343) is incorporated herein by reference inits entirety.

5.4.4 Immunomodulatory Agents and Anti-Inflammatory Agents

The method of the present invention provides methods of treatment forautoimmune diseases and inflammatory diseases comprising administrationof the antibodies of the present invention in conjunction with othertreatment agents. Examples of immunomodulatory agents include, but arenot limited to, methothrexate, ENBREL, REMICADE™, leflunomide,cyclophosphamide, cyclosporine A, and macrolide antibiotics (e.g., FK506(tacrolimus)), methylprednisolone (MP), corticosteroids, steriods,mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators.

Anti-inflammatory agents have exhibited success in treatment ofinflammatory and autoimmune disorders and are now a common and astandard treatment for such disorders. Any anti-inflammatory agentwell-known to one of skill in the art can be used in the methods of theinvention. Non-limiting examples of anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™ NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxgenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

5.4.5 Anti-Cancer Agents and Therapeutic Antibodies

In a specific embodiment, the methods of the invention encompass theadministration of one or more angiogenesis inhibitors such as but notlimited to: Angiostatin (plasminogen fragment); antiangiogenicantithrombin III; Angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab;BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complementfragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagenXVIII fragment); EGFr blockers/inhibitors (Iressa®, Tarceva®, Erbitux®,and ABX-EGF); Fibronectin fragment; Gro-beta; Halofuginone; Heparinases;Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin(hCG); IM-862; Interferon alpha/beta/gamma; Interferon inducible protein(IP-10); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat;Metalloproteinase inhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS27023A); MoAb IMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placentalribonuclease inhibitor; Plasminogen activator inhibitor; Plateletfactor-4 (PF4); Prinomastat; Prolactin 16 kD fragment;Proliferin-related protein (PRP); PTK 787/ZK 222594; Retinoids;Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248;Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1(TSP-1); TNP-470; Transforming growth factor-beta (TGF-β);Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD 6474;farnesyl transferase inhibitors (FTI); and bisphosphonates.

Anti-cancer agents that can be used in combination with antibodies ofthe invention in the various embodiments of the invention, includingpharmaceutical compositions and dosage forms and kits of the invention,include, but are not limited to: acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used in methods of theinvention include but are not limited to HERCEPTIN® (Trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer; REOPRO®(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection; PANOREX™ (edrecolomab)which is a murine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); Erbitux® (cetuximab) which is a chimericanti-EGFR IgG antibody (ImClone System); VITAXIN™ which is a humanizedanti-αVβ3 integrin antibody (Applied Molecular Evolution/Medlmmune);Campath 1H/LDP-03 which is a humanized anti CD52 IgG1 antibody(Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody(Protein Design Lab/Kanebo); RITUXAN™ (rituximab) which is a chimericanti-CD20 IgG1 antibody (DEC Pharm/Genentech, Roche/Zettyaku);LYIVIPHOCIDE™ (epratuzumab) which is a humanized anti-CD22 IgG antibody(Immunomedics); ICM3 which is a humanized anti-ICAM3 antibody (ICOSPharm); IDEC-114 which is a primatied anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ which is a radiolabelled murine anti-CD20antibody (IDEC/Schering AG); IDEC-131 which is a humanized anti-CD40Lantibody (IDEC/Eisai); IDEC-151 which is a primatized anti-CD4 antibody(IDEC); IDEC-152 which is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 which is a humanized anti-CD3 IgG(Protein Design Lab); 5G1.1 which is a humanized anti-complement factor5 (C5) antibody (Alexion Pharm); Humira® which is a human anti-TNF-αantibody (Abbott Laboratories); CDP870 which is a humanized anti-TNF-αFab fragment (Celltech); IDEC-151 which is a primatized anti-CD4 IgG1antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 which is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 which is ahumanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 which is ahumanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4Awhich is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™which is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ whichis a humanized anti-VLA-4 IgG antibody (Elan); and CAT-152 which is ahuman anti-TGF-β₂ antibody (Cambridge Ab Tech).

Other examples of therapeutic antibodies that can be used in combinationwith the antibodies of the invention are presented in Table 7.

TABLE 7 Monoclonal antibodies for Cancer Therapy that can be used incombination with the antibodies of the invention. Company ProductDisease Target Abgenix ABX-EGF Cancer EGF receptor AltaRex OvaRexovarian cancer tumor antigen CA125 BravaRex metastatic tumor antigenMUC1 cancers Antisoma Theragyn ovarian cancer PEM antigen(pemtumomabytrrium- 90) Therex breast cancer PEM antigen Boehringerblvatuzumab head & neck CD44 Ingelheim cancer Centocor/J&J PanorexColorectal 17-1A cancer ReoPro PTCA gp IIIb/IIIa ReoPro Acute MI gpIIIb/IIIa ReoPro Ischemic stroke gp IIIb/IIIa Corixa Bexocar NHL CD20CRC Technology MAb, idiotypic 105AD7 colorectal cancer gp72 vaccineCrucell Anti-EpCAM cancer Ep-CAM Cytoclonal MAb, lung cancer non-smallcell NA lung cancer Genentech Herceptin metastatic breast HER-2 cancerHerceptin early stage HER-2 breast cancer Rituxan Relapsed/refractoryCD20 low-grade or follicular NHL Rituxan intermediate & CD20 high-gradeNHL MAb-VEGF NSCLC, VEGF metastatic MAb-VEGF Colorectal VEGF cancer,metastatic AMD Fab age-related CD18 macular degeneration E-26 (2^(nd)gen. IgE) allergic asthma IgE & rhinitis IDEC Zevalin (Rituxan + lowgrade of CD20 yttrium-90) follicular, relapsed or refractory,CD20-positive, B-cell NHL and Rituximab- refractory NHL ImCloneCetuximab + innotecan refractory EGF receptor colorectal carcinomaCetuximab + cisplatin & newly diagnosed EGF receptor radiation orrecurrent head & neck cancer Cetuximab + newly diagnosed EGF receptorgemcitabine metastatic pancreatic carcinoma Cetuximab + cisplatin +recurrent or EGF receptor 5FU or Taxol metastatic head & neck cancerCetuximab + newly diagnosed EGF receptor carboplatin + paclitaxelnon-small cell lung carcinoma Cetuximab + cisplatin head & neck EGFreceptor cancer (extensive incurable local- regional disease & distantmetasteses) Cetuximab + radiation locally advanced EGF receptor head &neck carcinoma BEC2 + Bacillus small cell lung mimics gangliosideCalmette Guerin carcinoma GD3 BEC2 + Bacillus melanoma mimicsganglioside Calmette Guerin GD3 IMC-1C11 colorectal cancer VEGF-receptorwith liver metasteses ImmonoGen nuC242-DM1 Colorectal, nuC242 gastric,and pancreatic cancer ImmunoMedics LymphoCide Non-Hodgkins CD22 lymphomaLymphoCide Y-90 Non-Hodgkins CD22 lymphoma CEA-Cide metastatic solid CEAtumors CEA-Cide Y-90 metastatic solid CEA tumors CEA-Scan (Tc-99m-colorectal cancer CEA labeled arcitumomab) (radioimaging) CEA-Scan(Tc-99m- Breast cancer CEA labeled arcitumomab) (radioimaging) CEA-Scan(Tc-99m- lung cancer CEA labeled arcitumomab) (radioimaging) CEA-Scan(Tc-99m- intraoperative CEA labeled arcitumomab) tumors (radioimaging)LeukoScan (Tc-99m- soft tissue CEA labeled sulesomab) infection(radioimaging) LymphoScan (Tc-99m- lymphomas CD22 labeled)(radioimaging) AFP-Scan (Tc-99m- liver 7 gem-cell AFP labeled) cancers(radioimaging) Intracel HumaRAD-HN head & neck NA (+yttrium-90) cancerHumaSPECT colorectal NA imaging Medarex MDX-101 (CTLA-4) Prostate andCTLA-4 other cancers MDX-210 (her-2 Prostate cancer HER-2overexpression) MDX-210/MAK Cancer HER-2 MedImmune Vitaxin Cancer αvβ₃Merck KGaA MAb 425 Various cancers EGF receptor IS-IL-2 Various cancersEp-CAM Millennium Campath chronic CD52 (alemtuzumab) lymphocyticleukemia NeoRx CD20-streptavidin Non-Hodgkins CD20 (+biotin-yttrium 90)lymphoma Avidicin (albumin + metastatic NA NRLU13) cancer PeregrineOncolym (+iodine-131) Non-Hodgkins HLA-DR 10 beta lymphoma Cotara(+iodine-131) unresectable DNA-associated malignant proteins gliomaPharmacia C215 (+staphylococcal pancreatic NA Corporation enterotoxin)cancer MAb, lung/kidney lung & kidney NA cancer cancer nacolomabtafenatox colon & NA (C242 + staphylococcal pancreatic enterotoxin)cancer Protein Design Nuvion T cell CD3 Labs malignancies SMART M195 AMLCD33 SMART 1D10 NHL HLA-DR antigen Titan CEAVac colorectal CEA cancer,advanced TriGem metastatic GD2-ganglioside melanoma & small cell lungcancer TriAb metastatic breast MUC-1 cancer Trilex CEAVac colorectal CEAcancer, advanced TriGem metastatic GD2-ganglioside melanoma & small celllung cancer TriAb metastatic breast MUC-1 cancer Viventia BiotechNovoMAb-G2 Non-Hodgkins NA radiolabeled lymphoma Monopharm C colorectal& SK-1 antigen pancreatic carcinoma GlioMAb-H (+gelonin gliorna, NAtoxin) melanoma & neuroblastoma Xoma Rituxan Relapsed/refractory CD20low-grade or follicular NHL Rituxan intermediate & CD20 high-grade NHLING-1 adenomcarcinoma Ep-CAM

5.4.6 Vaccine Therapy

The invention provides a method for enhancing an immune response to avaccine composition in a subject, said method comprising administeringto said subject an antibody or a fragment thereof that specificallybinds FcγRIIB with greater affinity than said antibody or a fragmentthereof binds FcγRIIA, and a vaccine composition, wherein said antibodyor a fragment thereof enhances the immune response to said vaccinecomposition. In one particular embodiment, said antibody or a fragmentthereof enhances the immune response to said vaccine composition byenhancing antigen presentation/and or antigen processing of the antigento which the vaccine is directed at. Any vaccine composition known inthe art is useful in combination with the antibodies or fragmentsthereof of the invention.

In one embodiment, the invention encompasses the use of the antibodiesof the invention in combination with any cancer vaccine known in theart, e.g., Canvaxin™ (Cancer Vax, Corporation, melanoma and coloncancer); Oncophage (HSPPC-96; Antigenics; metastatic melanoma);HER-2/neu cancer vaccine, etc. The cancer vaccines used in the methodsand compositions of the invention can be, for example, antigen-specificvaccines, anti-idiotypic vaccines, dendritic cell vaccines, or DNAvaccines. The invention encompasses the use of the antibodies of theinvention with cell-based vaccines as described by Segal et al. (U.S.Pat. No. 6,403,080), which is incorporated herein by reference in itsentirety. The cell based vaccines used in combination with theantibodies of the invention can be either autologous or allogeneic.Briefly, the cancer-based vaccines as described by Segal et al. arebased on Opsonokine™ product by Genitrix, LLC. Opsonokines™ aregenetically engineered cytokines that, when mixed with tumor cells,automatically attach to the surface of the cells. When the “decorated”cells are administered as a vaccine, the cytokine on the cells activatescritical antigen presenting cells in the recipient, while also allowingthe antigen presenting cells to ingest the tumor cells. The antigenpresenting cells are then able to instruct “killer” T cells to find anddestroy similar tumor cells throughout the body. Thus, the Opsonokine™product converts the tumor cells into a potent anti-tumorimmunotherapeutic.

In one embodiment, the invention encompasses the use of the antibodiesof the invention in combination with any allergy vaccine known in theart. The antibodies of the invention, can be used, for example, incombination with recombinant hybrid molecules coding for the majortimothy grass pollen allergens used for vaccination against grass pollenallergy, as described by Linhart et al. (2000, FASEB Journal,16(10):1301-3, which is incorporated by reference). In addition theantibodies of the invention can be used in combination with DNA-basedvaccinations described by Homer et al. (2002, Allergy, 57 Suppl,72:24-9, which is incorporated by reference). Antibodies of theinvention can be used in combination with Bacille Clamett-Guerin (“BCG”)vaccination as described by Choi et al. (2002, Ann. Allergy AsthmaImmunology, 88(6): 584-91) and Barlan et al. (2002, Journal Asthma,39(3):239-46), both of which are incorporated herein by reference inentirety, to downregulate IgE secretion. The antibodies of the inventionare useful in treating food allergies. In particular the antibodies ofthe invention can be used in combination with vaccines or otherimmunotherapies known in the art (see Hourihane et al., 2002, Curr.Opin. Allergy Clin. Immunol. 2(3):227-31) for the treatment of peanutallergies.

The methods and compositions of the invention can be used in combinationwith vaccines, in which immunity for the antigen(s) is desired. Suchantigens may be any antigen known in the art. The antibodies of theinvention can be used to enhance an immune response, for example, toinfectious agents, diseased or abnormal cells such as, but not limitedto, bacteria (e.g., gram positive bacteria, gram negative bacteria,aerobic bacteria, Spirochetes, Mycobacteria, Rickettsias, Chlamydias,etc.), parasites, fungi (e.g., Candida albicans, Aspergillus, etc.),viruses (e.g., DNA viruses, RNA viruses, etc.), or tumors. Viralinfections include, but are not limited to, human immunodeficiency virus(HIV); hepatitis A virus, hepatitis B virus, hepatitis C virus,hepatitis D virus, or other hepatitis viruses; cytomagaloviruses, herpessimplex virus-1 (-2, -3, -4, -5, -6), human papilloma viruses;Respiratory syncytial virus (RSV), Parainfluenza virus (PIV), EpsteinBarr virus, human metapneumovirus (HMPV), influenza virus, Severe AcuteRespiratory Syndrome(SARS) or any other viral infections.

The invention encompasses methods and vaccine compositions comprisingcombinations of an antibody of the invention, an antigen and a cytokine.Preferably, the cytokine is IL-4, IL-10, or TGF-β.

The invention also encompasses the use of the antibodies of theinvention to enhance a humoral and/or cell mediated response against theantigen(s) of the vaccine composition. The invention further encompassesthe use of the antibodies of the invention to either prevent or treat aparticular disorder, where an enhanced immune response against aparticular antigen or antigens is effective to treat or prevent thedisease or disorder. Such diseases and disorders include, but are notlimited to, viral infections, such as HIV, CMV, hepatitis, herpes virus,measles, etc., bacterial infections, fungal and parasitic infections,cancers, and any other disease or disorder amenable to treatment orprevention by enhancing an immune response against a particular antigenor antigens.

5.5 Compositions and Methods of Administering

The invention provides methods and pharmaceutical compositionscomprising antibodies of the invention. The invention also providesmethods of treatment, prophylaxis, and amelioration of one or moresymptoms associated with a disease, disorder or infection byadministering to a subject an effective amount of a fusion protein or aconjugated molecule of the invention, or a pharmaceutical compositioncomprising a fusion protein or conjugated molecules of the invention. Ina preferred aspect, an antibody or fusion protein or conjugatedmolecule, is substantially purified (i.e., substantially free fromsubstances that limit its effect or produce undesired side-effects). Ina specific embodiment, the subject is an animal, preferably a mammalsuch as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.)and a primate (e.g., monkey such as, a cynomolgous monkey and a human).In a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer acomposition comprising antibodies of the invention, e.g., encapsulationin liposomes, microparticles, microcapsules, recombinant cells capableof expressing the antibody or fusion protein, receptor-mediatedendocytosis (See, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432),construction of a nucleic acid as part of a retroviral or other vector,etc.

In some embodiments, the antibodies of the invention are formulated inliposomes for targeted delivery of the antibodies of the invention.Liposomes are vesicles comprised of concentrically ordered phopsholipidbilayers which encapsulate an aqueous phase. Liposomes typicallycomprise various types of lipids, phospholipids, and/or surfactants. Thecomponents of liposomes are arranged in a bilayer configuration, similarto the lipid arrangement of biological membranes. Liposomes areparticularly preferred delivery vehicles due, in part, to theirbiocompatibility, low immunogenicity, and low toxicity. Methods forpreparation of liposomes are known in the art and are encompassed withinthe invention, see, e.g., Epstein et al., 1985, Proc. Natl. Acad. Sci.USA, 82: 3688; Hwang et al., 1980 Proc. Natl. Acad. Sci. USA, 77:4030-4; U.S. Pat. Nos. 4,485,045 and 4,544,545; all of which areincorporated herein by reference in their entirety.

The invention also encompasses methods of preparing liposomes with aprolonged serum half-life, i.e., enhanced circulation time, such asthose disclosed in U.S. Pat. No. 5,013,556. Preferred liposomes used inthe methods of the invention are not rapidly cleared from circulation,i.e., are not taken up into the mononuclear phagocyte system (MPS). Theinvention encompasses sterically stabilized liposomes which are preparedusing common methods known to one skilled in the art. Although notintending to be bound by a particular mechanism of action, stericallystabilized liposomes contain lipid components with bulky and highlyflexible hydrophilic moieties, which reduces the unwanted reaction ofliposomes with serum proteins, reduces oposonization with serumcomponents and reduces recognition by MPS. Sterically stabilizedliposomes are preferably prepared using polyethylene glycol. Forpreparation of liposomes and sterically stabilized liposome see, e.g.,Bendas et al., 2001 BioDrugs, 15(4): 215-224; Allen et al., 1987 FEBSLett. 223: 42-6; Klibanov et al., 1990 FEBS Lett., 268: 235-7; Blum etal., 1990, Biochim. Biophys. Acta., 1029: 91-7; Torchilin. et al., 1996,J. Liposome Res. 6: 99-116; Litzinger et al., 1994, Biochim. Biophys.Acta, 1190: 99-107; Maruyama et al., 1991, Chem. Pharm. Bull., 39:1620-2; Klibanov et al., 1991, Biochim Biophys Acta, 1062; 142-8; Allenet al., 1994, Adv. Drug Deliv. Rev, 13: 285-309; all of which areincorporated herein by reference in their entirety. The invention alsoencompasses liposomes that are adapted for specific organ targeting,see, e.g., U.S. Pat. No. 4,544,545, or specific cell targeting, see,e.g., U.S. Patent Application Publication No. 2005/0074403. Particularlyuseful liposomes for use in the compositions and methods of theinvention can be generated by reverse phase evaporation method with alipid composition comprising phosphatidylcholine, cholesterol, and PEGderivatized phosphatidylethanolamine (PEG-PE). Liposomes are extrudedthrough filters of defined pore size to yield liposomes with the desireddiameter. In some embodiments, a fragment of an antibody of theinvention, e.g., F(ab′), may be conjugated to the liposomes usingpreviously described methods, see, e.g., Martin et al., 1982, J. Biol.Chem. 257: 286-288, which is incorporated herein by reference in itsentirety.

The antibodies of the invention may also be formulated asimmunoliposomes. Immunoliposomes refer to a liposomal composition,wherein an antibody of the invention or a fragment thereof is linked,covalently or non-covalently to the liposomal surface. The chemistry oflinking an antibody to the liposomal surface is known in the art andencompassed within the invention, see, e.g., U.S. Pat. No. 6,787,153;Allen et al., 1995, Stealth Liposomes, Boca Rotan: CRC Press, 233-44;Hansen et al., 1995, Biochim. Biophys. Acta, 1239: 133-44; which areincorporated herein by reference in their entirety. In most preferredembodiments, immunoliposomes for use in the methods and compositions ofthe invention are further sterically stabilized. Preferably, theantibodies of the invention are linked covalently or non-covalently to ahydrophobic anchor, which is stably rooted in the lipid bilayer of theliposome. Examples of hydrophobic anchors include but are not limited tophospholipids, e.g., phosoatidylethanolamine (PE), phospahtidylinositol(PI). To achieve a covalent linkage between an antibody and ahydrophobic anchor, any of the known biochemical strategies in the artmay be used, see, e.g., J. Thomas August, ed., 1997, Gene Therapy:Advances in Pharmacology, Volume 40, Academic Press, San Diego, Calif.,p. 399-435, which is incorporated herein by reference in its entiretyFor example, a functional group on an antibody molecule may react withan active group on a liposome associated hydrophobic anchor, e.g., anamino group of a lysine side chain on an antibody may be coupled toliposome associated N-glutaryl-phosphatidylethanolamine activated withwater-soluble carbodiimide; or a thiol group of a reduced antibody canbe coupled to liposomes via thiol reactive anchors such aspyridylthiopropionyl-phosphatidylethanolamine. See, e.g., Dietrich etal., 1996, Biochemistry, 35: 1100-1105; Loughrey et al., 1987, Biochim.Biophys. Acta, 901: 157-160; Martin et al., 1982, J. Biol. Chem. 257:286-288; Martin et al., 1981, Biochemistry, 20: 4429-38; all of whichare incorporated herein by reference in their entirety. Although notintending to be bound by a particular mechanism of action,immunoliposomal formulations comprising an antibody of the invention areparticularly effective as therapeutic agents, since they deliver theantibody to the cytoplasm of the target cell, i.e., the cell comprisingthe FcγRIIB receptor to which the antibody binds. The immunoliposomespreferably have an increased half-life in blood, specifically targetcells, and can be internalized into the cytoplasm of the target cellsthereby avoiding loss of the therapeutic agent or degradation by theendolysosomal pathway.

The invention encompasses immunoliposomes comprising an antibody of theinvention or a fragment thereof. In some embodiments, theimmunoliposomes further comprise one or more additional therapeuticagents, such as those disclosed herein.

The immunoliposomal compositions of the invention comprise one or morevesicle forming lipids, an antibody of the invention or a fragment orderivative thereof, and optionally a hydrophilic polymer. A vesicleforming lipid is preferably a lipid with two hydrocarbon chains, such asacyl chains and a polar head group. Examples of vesicle forming lipidsinclude phospholipids, e.g., phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol,sphingomyelin, and glycolipids, e.g., cerebrosides, gangliosides.Additional lipids useful in the formulations of the invention are knownto one skilled in the art and encompassed within the invention. In someembodiments, the immunoliposomal compositions further comprise ahydrophilic polymer, e.g., polyethylene glycol, and ganglioside GM1,which increases the serum half life of the liposome. Methods ofconjugating hydrophilic polymers to liposomes are well known in the artand encompassed within the invention. For a review of immunoliposomesand methods of preparing them, see, e.g., U.S. Patent ApplicationPublication No. 2003/0044407; PCT International Publication No. WO97/38731, Vingerhoeads et al., 1994, Immunomethods, 4: 259-72; Maruyama,2000, Biol. Pharm. Bull. 23(7): 791-799; Abra et al., 2002, Journal ofLiposome Research, 12(1&2): 1-3; Park, 2002, Bioscience Reports, 22(2):267-281; Bendas et al., 2001 BioDrugs, 14(4): 215-224, J. Thomas August,ed., 1997, Gene Therapy: Advances in Pharmacology, Volume 40, AcademicPress, San Diego, Calif., p. 399-435, all of which are incorporatedherein by reference in their entireties.

Methods of administering an antibody of the invention include, but arenot limited to, parenteral administration (e.g., intradermal,intramuscular, intraperitoneal, intravenous and subcutaneous), epidural,and mucosal (e.g., intranasal and oral routes). In a specificembodiment, the antibodies of the invention are administeredintramuscularly, intravenously, or subcutaneously. The compositions maybe administered by any convenient route, for example, by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985, 20; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety.

The invention also provides that the antibodies of the invention arepackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity of antibody. In one embodiment, theantibodies of the invention are supplied as a dry sterilized lyophilizedpowder or water free concentrate in a hermetically sealed container andcan be reconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject. Preferably, theantibodies of the invention are supplied as a dry sterile lyophilizedpowder in a hermetically sealed container at a unit dosage of at least 5mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, atleast 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. Thelyophilized antibodies of the invention should be stored at between 2and 8° C. in their original container and the antibodies should beadministered within 12 hours, preferably within 6 hours, within 5 hours,within 3 hours, or within 1 hour after being reconstituted. In analternative embodiment, antibodies of the invention are supplied inliquid form in a hermetically sealed container indicating the quantityand concentration of the antibody, fusion protein, or conjugatedmolecule. Preferably, the liquid form of the antibodies are supplied ina hermetically sealed container at least 1 mg/ml, more preferably atleast 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml,at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100mg/ml, at least 150 mg/ml, at least 200 mg/ml of the antibodies.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For antibodies encompassed by the invention, the dosage administered toa patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. Generally, humanantibodies have a longer half-life within the human body than antibodiesfrom other species due to the immune response to the foreignpolypeptides. Thus, lower dosages of human antibodies and less frequentadministration is often possible. Further, the dosage and frequency ofadministration of antibodies of the invention or fragments thereof maybe reduced by enhancing uptake and tissue penetration of the antibodiesby modifications such as, for example, lipidation.

In one embodiment, the dosage of the antibodies of the inventionadministered to a patient are 0.01 mg to 1000 mg/day, when used assingle agent therapy. In another embodiment the antibodies of theinvention are used in combination with other therapeutic compositionsand the dosage administered to a patient are lower than when saidantibodies are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering an antibody of the invention, care must be taken touse materials to which the antibody or the fusion protein does notabsorb.

In another embodiment, the compositions can be delivered in a vesicle,in particular a liposome (See Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.).

In yet another embodiment, the compositions can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more antibodies of the invention. See,e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCTpublication WO 96/20698; Ning et al., 1996, “IntratumoralRadioimmunotheraphy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.,1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al., 1997, “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety. In one embodiment, apump may be used in a controlled release system (See Langer, supra;Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980,Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574). Inanother embodiment, polymeric materials can be used to achievecontrolled release of antibodies (see e.g., Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger andPeppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; See alsoLevy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos.5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT PublicationNo.

WO 99/15154; and PCT Publication No. WO 99/20253). Examples of polymersused in sustained release formulations include, but are not limited to,poly(-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target (e.g., the lungs), thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).In another embodiment, polymeric compositions useful as controlledrelease implants are used according to Dunn et al. (See U.S. Pat. No.5,945,155). This particular method is based upon the therapeutic effectof the in situ controlled release of the bioactive material from thepolymer system. The implantation can generally occur anywhere within thebody of the patient in need of therapeutic treatment. In anotherembodiment, a non-polymeric sustained delivery system is used, whereby anon-polymeric implant in the body of the subject is used as a drugdelivery system. Upon implantation in the body, the organic solvent ofthe implant will dissipate, disperse, or leach from the composition intosurrounding tissue fluid, and the non-polymeric material will graduallycoagulate or precipitate to form a solid, microporous matrix (See U.S.Pat. No. 5,888,533).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698;Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al.,1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397;Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.Bioact. Mater. 24:759-760, each of which is incorporated herein byreference in its entirety.

In a specific embodiment where the composition of the invention is anucleic acid encoding an antibody, the nucleic acid can be administeredin vivo to promote expression of its encoded antibody, by constructingit as part of an appropriate nucleic acid expression vector andadministering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

For antibodies, the therapeutically or prophylactically effective dosageadministered to a subject is typically 0.1 mg/kg to 200 mg/kg of thesubject's body weight. Preferably, the dosage administered to a subjectis between 0.1 mg/kg and 20 mg/kg of the subject's body weight and morepreferably the dosage administered to a subject is between 1 mg/kg to 10mg/kg of the subject's body weight. The dosage and frequency ofadministration of antibodies of the invention may be reduced also byenhancing uptake and tissue penetration (e.g., into the lung) of theantibodies or fusion proteins by modifications such as, for example,lipidation.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of antibodies of the invention can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with antibodies of the inventionin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In other embodiments, the pharmaceuticalcompositions of the invention are administered once a day, twice a day,or three times a day. In other embodiments, the pharmaceuticalcompositions are administered once a week, twice a week, once every twoweeks, once a month, once every six weeks, once every two months, twicea year or once per year. It will also be appreciated that the effectivedosage of the antibodies used for treatment may increase or decreaseover the course of a particular treatment.

5.5.1 Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of antibodies of the invention and apharmaceutically acceptable carrier.

In one particular embodiment, the pharmaceutical composition comprisesof a therapeutically effective amount of an antibody or a fragmentthereof that binds FcγRIIB with a greater affinity than said antibody ora fragment thereof binds FcγRIIA, a cytotoxic antibody that specificallybinds a cancer antigen, and a pharmaceutically acceptable carrier. Inanother embodiment, said pharmaceutical composition further comprisesone or more anti-cancer agents.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limited tothose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The present invention also provides pharmaceutical compositions and kitscomprising a FcγRIIB antagonist for use in the prevention, treatment,management, or amelioration of a B-cell malignancy, or one or moresymptoms thereof. In particular, the present invention providespharmaceutical compositions and kits comprising a FcγRIIB antagonist, ananalog, derivative or an anti-FcγRIIB antibody or an antigen-bindingfragment thereof.

5.5.2 Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or fusion proteins, are administered to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, Science 260:926-932 (1993); Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215; and Scholl,2003, J. Biomed Biotechnol 2003:35-47. Methods commonly known in the artof recombinant DNA technology which can be used are described in Ausubelet al. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons, N Y (1993); and Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises nucleicacids encoding an antibody, said nucleic acids being part of anexpression vector that expresses the antibody in a suitable host. Inparticular, such nucleic acids have promoters, preferably heterologouspromoters, operably linked to the antibody coding region, said promoterbeing inducible or constitutive, and, optionally, tissue-specific. Inanother particular embodiment, nucleic acid molecules are used in whichthe antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller and Smithies, 1989, Proc.Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989, Nature342:435-438).

In another preferred aspect, a composition of the invention comprisesnucleic acids encoding a fusion protein, said nucleic acids being a partof an expression vector that expression the fusion protein in a suitablehost. In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the coding region of a fusionprotein, said promoter being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the coding sequence of the fusion proteinand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the fusion protein encoding nucleicacids.

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or by coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (See, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (See, e.g., U.S. Patent Application Publication No.2005/0002903; PCT Publications WO 92/06180; WO 92/22635; WO92/20316;WO93/14188; WO 93/20221). Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989,Nature 342:43 5-438).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody or a fusion protein are used. Forexample, a retroviral vector can be used (See Miller et al., 1993, Meth.Enzymol. 217:581-599). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyor a fusion protein to be used in gene therapy are cloned into one ormore vectors, which facilitates delivery of the nucleotide sequence intoa subject. More detail about retroviral vectors can be found in Boesenet al., (1994, Biotherapy 6:291-302), which describes the use of aretroviral vector to deliver the mdr 1 gene to hematopoietic stem cellsin order to make the stem cells more resistant to chemotherapy. Otherreferences illustrating the use of retroviral vectors in gene therapyare: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Klein et al.,1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics andDevel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson (CurrentOpinion in Genetics and Development 3:499-503, 1993, present a review ofadenovirus-based gene therapy. Bout et al., (Human Gene Therapy, 5:3-10,1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang et al., 1995, Gene Therapy 2:775-783. In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (see, e.g., Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.204:289-300 and U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to, transfection, electroporation,microinjection, infection with a viral or bacteriophage vector,containing the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcellmediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (See, e.g., Loeffler and Behr, 1993, Meth. Enzymol.217:599-618, Cohen et al., 1993, Meth. Enzymol. 217:618-644; and Clin.Pharma. Ther. 29:69-92, 1985) and may be used in accordance with thepresent invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody or a fusion protein areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (See e.g., PCT Publication WO94/08598; Stemple and Anderson, 1992, Cell 7 1:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

5.5.3 Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with antibodies of the invention. Additionally,one or more other prophylactic or therapeutic agents useful for thetreatment of a disease can also be included in the pharmaceutical packor kit. The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more antibodies ofthe invention. In another embodiment, a kit further comprises one ormore other prophylactic or therapeutic agents useful for the treatmentof cancer, in one or more containers. In another embodiment, a kitfurther comprises one or more cytotoxic antibodies that bind one or morecancer antigens associated with cancer. In certain embodiments, theother prophylactic or therapeutic agent is a chemotherapeutic. In otherembodiments, the prophylactic or therapeutic agent is a biological orhormonal therapeutic.

5.6 Characterization and Demonstration of Therapeutic Utility

Several aspects of the pharmaceutical compositions or prophylactic ortherapeutic agents of the invention are preferably tested in vitro,e.g., in a cell culture system, and then in vivo, e.g., in an animalmodel organism, such as a rodent animal model system, for the desiredtherapeutic activity prior to use in humans. For example, assays whichcan be used to determine whether administration of a specificpharmaceutical composition is indicated, include cell culture assays inwhich a patient tissue sample is grown in culture, and exposed to orotherwise contacted with a pharmaceutical composition, and the effect ofsuch composition upon the tissue sample is observed, e.g., inhibition ofor decrease in growth and/or colony formation in soft agar or tubularnetwork formation in three-dimensional basement membrane orextracellular matrix preparation. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective prophylactic or therapeutic molecule(s)for each individual patient. Alternatively, instead of culturing cellsfrom a patient, therapeutic agents and methods may be screened usingcells of a tumor or malignant cell line. In various specificembodiments, in vitro assays can be carried out with representativecells of cell types involved in an autoimmune or inflammatory disorder(e.g., T cells), to determine if a pharmaceutical composition of theinvention has a desired effect upon such cell types. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc. Additional assays include raftassociation, CDC, ADCC and apoptosis assays as known in the art anddescribed in the Examples.

Combinations of prophylactic and/or therapeutic agents can be tested insuitable animal model systems prior to use in humans. Such animal modelsystems include, but are not limited to, rats, mice, chicken, cows,monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in theart may be used. In a specific embodiment of the invention, combinationsof prophylactic and/or therapeutic agents are tested in a mouse modelsystem. Such model systems are widely used and well-known to the skilledartisan. Prophylactic and/or therapeutic agents can be administeredrepeatedly. Several aspects of the procedure may vary such as thetemporal regime of administering the prophylactic and/or therapeuticagents, and whether such agents are administered separately or as anadmixture.

Preferred animal models for use in the methods of the invention are forexample, transgenic mice expressing FcγR on mouse effector cells, e.g.,any mouse model described in U.S. Pat. No. 5,877,396 (which isincorporated herein by reference in its entirety). Transgenic mice foruse in the methods of the invention include but are not limited to micecarrying human FcγRIIIA, mice carrying human FcγRIIA, mice carryinghuman FcγRIIB and human FcγRIIIA, mice carrying human FcγRIIB and humanFcγRIIA.

Once the prophylactic and/or therapeutic agents of the invention havebeen tested in an animal model they can be tested in clinical trials toestablish their efficacy. Establishing clinical trials will be done inaccordance with common methodologies known to one skilled in the art,and the optimal dosages and routes of administration as well as toxicityprofiles of the compositions of the invention can be established usingroutine experimentation.

The anti-inflammatory activity of the combination therapies of inventioncan be determined by using various experimental animal models ofinflammatory arthritis known in the art and described in Crofford L. J.and Wilder R. L., “Arthritis and Autoimmunity in Animals”, in Arthritisand Allied Conditions: A Textbook of Rheumatology, McCarty et al.(eds.), Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneousanimal models of inflammatory arthritis and autoimmune rheumaticdiseases can also be used to assess the anti-inflammatory activity ofthe combination therapies of invention. The following are some assaysprovided as examples, and not by limitation.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis ratmodels, collagen-induced arthritis rat and mouse models andantigen-induced arthritis rat, rabbit and hamster models, all describedin Crofford L. J. and Wilder R. L., “Arthritis and Autoimmunity inAnimals”, in Arthritis and Allied Conditions: A Textbook ofRheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993),incorporated herein by reference in its entirety.

The anti-inflammatory activity of the combination therapies of inventioncan be assessed using a carrageenan-induced arthritis rat model.Carrageenan-induced arthritis has also been used in rabbit, dog and pigin studies of chronic arthritis or inflammation. Quantitativehistomorphometric assessment is used to determine therapeutic efficacy.The methods for using such a carrageenan-induced arthritis model isdescribed in Hansra P. et al., “Carrageenan-Induced Arthritis in theRat,” Inflammation, 24(2): 141-155, (2000). Also commonly used arezymosan-induced inflammation animal models as known and described in theart.

The anti-inflammatory activity of the combination therapies of inventioncan also be assessed by measuring the inhibition of carrageenan-inducedpaw edema in the rat, using a modification of the method described inWinter C. A. et al., “Carrageenan-Induced Edema in Hind Paw of the Ratas an Assay for Anti-inflammatory Drugs” Proc. Soc. Exp. Biol Med. 111,544-547, (1962). This assay has been used as a primary in vivo screenfor the anti-inflammatory activity of most NSAIDs, and is consideredpredictive of human efficacy. The anti-inflammatory activity of the testprophylactic or therapeutic agents is expressed as the percentinhibition of the increase in hind paw weight of the test group relativeto the vehicle dosed control group.

Additionally, animal models for inflammatory bowel disease can also beused to assess the efficacy of the combination therapies of invention(Kim et al., 1992, Scand. J. Gastroentrol. 27:529-537; Strober, 1985,Dig. Dis. Sci. 30(12 Suppl):3S-10S). Ulcerative cholitis and Crohn'sdisease are human inflammatory bowel diseases that can be induced inanimals. Sulfated polysaccharides including, but not limited toamylopectin, carrageen, amylopectin sulfate, and dextran sulfate orchemical irritants including but not limited to trinitrobenzenesulphonicacid (TNBS) and acetic acid can be administered to animals orally toinduce inflammatory bowel diseases.

Animal models for asthma can also be used to assess the efficacy of thecombination therapies of invention. An example of one such model is themurine adoptive transfer model in which aeroallergen provocation of TH1or TH2 recipient mice results in TH effector cell migration to theairways and is associated with an intense neutrophilic (TH1) andeosinophilic (TH2) lung mucosal inflammatory response (Cohn et al.,1997, J. Exp. Med. 1861737-1747).

Animal models for autoimmune disorders can also be used to assess theefficacy of the combination therapies of invention. Animal models forautoimmune disorders such as type 1 diabetes, thyroid autoimmunity,systemic lupus eruthematosus, and glomerulonephritis have been developed(Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999,Biochimie 81:511-515; Foster, 1999, Semin. Nephrol. 19:12-24).

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for autoimmune and/orinflammatory diseases.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in Relevanceof Tumor Models for Anticancer Drug Development (1999, eds. Fiebig andBurger); Contributions to Oncology (1999, Karger); The Nude Mouse inOncology Research (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher), herein incorporated by referencein their entireties.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer, inflammatory disorder, or autoimmune disease.

5.7 Diagnostic Methods

Labeled antibodies of the invention can be used for diagnostic purposesto detect, diagnose, or monitor diseases, disorders or infections. Theinvention provides for the detection or diagnosis of a disease, disorderor infection, particularly an autoimmune disease comprising: (a)assaying the expression of FcγRIIB in cells or a tissue sample of asubject using one or more antibodies that immunospecifically bind toFcγRIIB; and (b) comparing the level of the antigen with a controllevel, e.g., levels in normal tissue samples, whereby an increase in theassayed level of antigen compared to the control level of the antigen isindicative of the disease, disorder or infection.

Antibodies of the invention can be used to assay FcγRIIB levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al.,1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods usefulfor detecting protein gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (MA).Suitable antibody assay labels are known in the art and include enzymelabels, such as, alkaline phosphatase, glucose oxidase; radioisotopes,such as iodine (125I, ¹³¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹²¹In), and technetium (^(99m)Tc); luminescent labels, such asluminol; and fluorescent labels, such as fluorescein and rhodamine.

One aspect of the invention is the detection and diagnosis of a disease,disorder, or infection in a human. In one embodiment, diagnosiscomprises: a) administering (for example, parenterally, subcutaneously,or intraperitoneally) to a subject an effective amount of a labeledantibody that immunospecifically binds to FcγRIIB; b) waiting for a timeinterval following the administration for permitting the labeledantibody to preferentially concentrate at sites in the subject whereFcγRIIB is expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled antibody in the subject, such that detection of labeled antibodyabove the background level indicates that the subject has the disease,disorder, or infection. In accordance with this embodiment, the antibodyis labeled with an imaging moiety which is detectable using an imagingsystem known to one of skill in the art. Background level can bedetermined by various methods including, comparing the amount of labeledmolecule detected to a standard value previously determined for aparticular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibodywill then preferentially accumulate at the location of cells whichcontain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodiesand Their Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In one embodiment, monitoring of a disease, disorder or infection iscarried out by repeating the method for diagnosing the disease, disorderor infection, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

6. EXAMPLES 6.1 Preparation of Monoclonal Antibodies

A mouse monoclonal antibody was produced from clone 8B5.3.4 with ATCCaccession number PTA-7610. A mouse monoclonal antibody that specificallybinds FcγRIIB with greater affinity than said monoclonal antibody bindsFcγRIIA, was generated. Transgenic FcγRIIA mice (generated in Dr.Ravetch Laboratory, Rockefeller University) were immunized with FcγRIIBpurified from supernatant of 293 cells that had been transfected withcDNA encoding the extracellular domain of the human FcγRIIB receptor,residues 1-180. Hybridoma cell lines from spleen cells of these micewere produced and screened for antibodies that specifically bind FcγRIIBwith greater affinity than the antibodies bind FcγRIIA.

6.2 Antibody Screening and Characterization

6.2.1 Materials and Methods

Supernatants from hybridoma cultures are screened for immunoreactivityagainst FcγRIIA or FcγRIIB using ELISA assays. In each case, the plateis coated with 100 ng/well of FcγRIIA or FcγRIIB The binding of theantibody to the specific receptor is detected with goat anti-mouse HRPconjugated antibody by monitoring the absorbance at 650 nm.

In the blocking ELISA experiment, the ability of the antibody from thehybridoma supernatant to block binding of aggregated IgG to FcγRIIB ismonitored. The plate is blocked with the appropriate “blocking agent”,washed three times (200 μl/well) with wash buffer (PBS plus 0.1% Tween).The plate is pre-incubated with hybridoma supernatant for 1 hour at 37°C. Subsequent to blocking, a fixed amount of aggregated biotinylatedhuman IgG (1 μg/well) is added to the wells to allow the aggregate tobind to the FcγRIIB receptor. This reaction is carried out for two hoursat 37° C. Detection is then monitored, after additional washing, withstreptavidin horseradish peroxidase conjugate, which detects the boundaggregated IgG. The absorbance at 650 nm is proportional to the boundaggregated IgG.

In a β-hexoaminidase release assay the ability of an antibody from thehybridoma supernatant to inhibit Fcγ-induced release of β-hexoaminidaseis monitored. RBL-2H3 cells are transfected with human FcγRIIB; cellsare stimulated with various concentration of goat anti-mouse F(ab)₂fragment ranging from 0.03 μg/mL to 30 μg/mL; sensitized with eithermouse IgE alone (at 0.01 μg/mL) or with an anti-FcγRIIB antibody. After1 hour incubation at 37° temperature, the cells are spun down; thesupernatant is collected; and the cells are lysed. The β-hexoaminidaseactivity released in the supernatant is determined in a colorometricassay using p-nitrophenyl N-acetyl-β D-glucoasminide. The releaseβ-hexoaminidase activity is expressed as a percentage of the releasedactivity relative to the total activity.

BIAcore Analysis: Antibody binding to CD32A-H¹³¹, CD32A-R¹³¹ or CD32Bwas analyzed by surface plasmon resonance in a BIAcore 3000 biosensor(Biacore AB, Uppsala, Sweden) by using soluble extracellular domains ofthe receptors expressed in 293H cells. The capturing antibody, a F(ab′)₂fragment of a goat anti-mouse Fc-specific antibody (JacksonImmunoresearch, West Grove, Pa.), was immobilized on the CM-5 sensorchip according to the procedure recommended by the manufacturer.Briefly, the carboxyl groups on the sensor chip surface were activatedwith an injection of a solution containing 0.2M N-ethyl-N-(3dietylamino-propyl) carbodiimide and 0.05M N-hydroxy-succinimide. TheF(ab′)₂ fragment was then injected over the activated CM-5 surface in 10mM sodium-acetate, pH 5.0, at flow rate 5 μl/min for 420 sec, followedby 1 M ethanolamine for deactivation. Binding experiments were performedin HBS-P buffer containing 10 mM Hepes, pH 7.4, 150 mM NaCl, and 0.005%P20 surfactant. Each monoclonal antibody was captured on the CM-5 chipby injecting a 300 nM-antibody solution at flow rate 5 μl/min for 240sec, followed by an injection of the monomeric soluble receptors atconcentration of 100 nM and a flow rate 50 μl/min for 120 sec withdissociation time 180 sec. Regeneration of the F(ab′)₂ GAM surface wasperformed by pulse injection of 50 mM glycine, pH 1.5 and 50 mM NaOH.Reference curves were obtained by injection of each soluble receptorover the immobilized F(ab′)₂ GAM surface with no captured antibody.Reference curves were subtracted and responses were normalized to thesame level of captured antibody. To obtain kinetic parameters of bindingof soluble receptors to captured antibodies, binding curves tocorresponding IgG at corresponding concentrations were subtracted.Resulted curves were analyzed by separate ka/kd fit. K_(D) values werecalculated as average of four curves at two different concentrations.

FACS ANALYSIS: CHO cells, expressing FcγRIIB are stained with variousantibodies and analyzed by FACS. In one series of experiment, the cellsare directly labeled to determine if the monoclonal antibodies recognizethe receptor.

In the blocking FACS experiment, the ability of the antibody from thehybridoma supernatant to block the binding of aggregated IgG to FcγRIIBis monitored. About 1 million cells (CHO cells expressing FcγRIIB) foreach sample are incubated on ice for 30 minutes with 2 μg of the isotypecontrol (mouse IgG1) or with the 8B5.3.4 antibody. Cells are washed oncewith PBS+1% BSA and incubated with 1 μg of aggregated biotinylated humanIgG for 30 minutes on ice. Cells are washed and the secondary antibodiesare added, goat anti-mouse-FITC to detect the bound antibody andStreptavidin-PE conjugated to detect the bound aggregated biotinylatedhuman IgG and incubated on ice for 30 minutes. Cells are washed andanalyzed by FACS.

B Lymphocytes are stained to detect the presence of FcγRIIB and CD20.200 μl of “buffy coat” for each sample is incubated on ice with 2 μg ofisotype control or the monoclonal antibodies, 8B5.3.4. Cells are washedonce with PBS+1% BSA and incubated with 1 μl of goat anti mouse-PEantibody for 30 minutes on ice. Cells are washed once and CD20-FITCantibody (2 μg) is added to the samples and incubated on ice for 30minutes. All samples are washed with PBS+1% BSA once and the cells areanalyzed by FACS.

Human PBMCs are stained with 8B5.3.4 and IV.3 antibodies, followed by agoat anti-mouse-Cyanine (Cy5) conjugated antibody (two color stainingusing anti-CD20-FITC conjugated for B lymphocytes, anti-CD14-PEconjugated for monocytes, anti-CD56-PE conjugated for NK cells andanti-CD16-PE conjugated for granulocytes.

ADCC ASSAY: 4-5×10⁶ target cells expressing Her2/neu antigen (IGROV-1 orSKBR-3 cells) are labeled with bis(acetoxymethyl)2,2′:6′,2″-terpyridine-t-6″-dicarboxylate (DELFIA BATDA Reagent, PerkinElmer/Wallac). BATDA reagent is added to the cells and the mixture isincubated at 37° C. preferably under 5% CO₂, for at least 30 minutes.The cells are then washed with a physiological buffer, e.g., PBS with0.125 mM sulfinpyrazole, and media containing 0.125 mM sulfinpyrazole.The labeled target cells are added to effector cells, e.g., PBMC, toproduce effector:target ratios of approximately 50:1, 75:1, or 100:1.PBMC is isolated by layering whole blood onto Ficoll-Hypaque (Sigma) andspinning at room temperature for 30 mins at 500 g. The leukocyte layeris harvested as effectors for Europium-based ADCC assays. Frozen orfreshly isolated elutriated monocytes (Advanced Biotechnologies, MD) isused as effectors with the tumor target cell lines at varying effectorto target ratio of 100:1 to 10:1 and the concentration of the antibodiesis titrated from 1-15 μg/ml. Monocytes obtained as frozen stocksstimulated with cytokines is used as effector cells in ADCC assays. Iffrozen monocytes perform optimally they will be routinely used otherwisefresh cells will be used. MDM will be prepared by treatment withcytokines GM-CSF or M-CSF that are known to enhance the viability anddifferentiation of monocytes in culture. MDM will be stimulated withcytokines and the expression of the various FcγRs (I, IIA, IIB, andIIIA) determined by FACS analysis.

The effector and target cells are incubated for at least two hours, andup to 16 hours, at 37° C., under 5% CO₂ in the presence of an anti-tumorantibody, specific for an antigen expressed on the target cells,Her2/neu, and in the presence or absence of an anti-FcγRIIB antibody. Achimeric 4D5 antibody that has been engineered to contain the N297Amutation which is used as a negative control since this antibody bindsthe tumor target cells via its variable region. Loss of glycosylation atthis site abolishes binding of the Fc region of the antibody to FcγR.Commercially available human IgG1/k serves as an isotype control for theanti-FcγRIIB antibody. Cell supernatants are harvested and added to anacidic europium solution (e.g., DELFIA Europium Solution, PerkinElmer/Wallac). The fluorescence of the Europium-TDA chelates formed isquantitated in a time-resolved fluorometer (e.g., Victor 1420, PerkinElmer/Wallac). Maximal release (MR) and spontaneous release (SR) aredetermined by incubation of target cells with 1% TX-100 and media alone,respectively. Antibody independent cellular cytotoxicity (AICC) ismeasured by incubation of target and effector cells in the absence ofantibody. Each assay is preferably performed in triplicate. The meanpercentage specific lysis is calculated as: Experimental release(ADCC)−AICC)/(MR−SR)×100.

6.2.2 Characterization of the Monoclonal Antibody Produced from the8B5.3.4 Clone

Direct binding of the antibody produced from the 8B5.3.4 clone toFcγRIIA and FcγRIIB: The direct binding of the MAb 8B5.3.4 produced fromthe 8B5.3.4 clone to FcγRIIA and FcγRIIB was compared by ELISA assaywith FcγRIIA and FcγRIIB-coated plates (Table 8 and FIG. 1). Culturesupernatant from the 8B5.3.4 hybridoma clone containing the MAb 8B5.3.4was tested (in duplicate) for specific binding to FcγRIIA and FcγRIIBThe supernatant from hybridoma clone 8B5.3.4 has significantly higheraffinity for FcγRIIB than FcγRIIA, as indicated by the higher absorbancevalues relative to the positive and negative controls in Table 8 (FIG.1).

TABLE 8 Results of ELISA assay to characterize the binding of MAb8B5.3.4 produced by hybridoma clone 8B5.3.4 to FcγRIIA and FcγRIIB. Theabsorbance values (in duplicate) at 650 nm are reported for each sample.CD32A-coated wells CD32B- coated wells 8B5.3.4 0.094 2.044 0.092 2.028Negative Control 0.071 0.074 0.065 0.078 Positive Control 0.340 0.3320.352 0.308

Isotyping the MAb 8B5.3.4 produced from the 8B5.3.4 clone: The isotypeof the MAb 8B5.3.4 was determined by ELISA assay (Table 9 and FIG. 2).Antibodies against various isotypes indicated were assayed. Theabsorbance values in FIG. 2 show that the anti-IgG, anti-IgG1, andanti-kappa antibodies have the highest reactivity with the MAb 8B5.3.4,which indicates that the MAb 8B5.3.4 has an IgG1/kappa isotype.

TABLE 9 Results of ELISA assay to characterize the isotype of MAb8B5.3.4 produced by hybridoma clone 8B5.3.4. The absorbance values at650 nm are reported for the indicated antibody isotype. Antibody IsotypeAbsorbance @ 650 nm IgG 2.828 IgG3 0.165 IgM 0.105 IgG2a 0.246 IgG2b0.213 IgG1 2.488 IgA 0.363 kappa 0.511

6.2.3 In Vivo ADCC Assays

6.2.3.1 Activity of FcγRIIB Antibodies in Xenograft Murine Models UsingHuman Tumor Cell Lines

Six to eight week old female Balb/c nude mice (Jackson Laboratories, BarHarbor, Me.; Taconic) are utilized for establishing the xenograftovarian and breast carcinoma models. Mice are housed in BiosafetyLevel-2 facilities for the xenograft model using the ascites-derivedovarian cells and pleural effusion-derived breast cancer cells assources of tumors. Mice are placed in groups of 4 for these experimentsand monitored three times weekly. The weight of the mice and survivaltime are recorded and criteria for growing tumors is abdominaldistention and palpable tumors. Mice showing signs of visible discomfortor that reach 5 grams in tumor weight are euthanized with carbon dioxideand autopsied. The antibody-treated animals are placed under observationfor an additional two months after the control group.

Establishment of the xenograft tumor model with tumor cell lines. Inorder to establish the xenograft tumor model, 5×10⁶ viable IGROV-1 orSKBR-3 cells are injected s.c into three age and weight matched femalenude athymic mice with Matrigel (Becton Dickinson). The estimated weightof the tumor is calculated by the formula: length×(width)/2 not toexceed 3 grams. For in vivo passaging of cells for expansion,anchorage-dependent tumor is isolated and the cells dissociated byadding 1 μg of collagenase (Sigma) per gram of tumor at 37 C overnight.

Injection of IGROV-1 cells subcutaneously gives rise to fast growingtumors while the intraperitoneal route induces a peritonealcarcinomatosis which kills the mice in 2 months. Since the IGROV-1 cellsform tumors within 5 weeks, at day 1 after tumor cell injection,monocytes as effectors are co-injected i.p. along with therapeuticantibodies ch4D5 and ch8B5.3.4 at 4 μg each per gm of mouse body weight(mbw) (Table 10). The initial injection is followed by weekly injectionsof antibodies for 4-6 weeks thereafter. Human effectors cells arereplenished once in two weeks. A group of mice will receive notherapeutic antibody but will be injected with ch4D5 N297A and humanIgG1 as isotype control antibodies for the anti-tumor and ch8B5.3.4antibody, respectively.

TABLE 10 Outline for tumor clearance studies with chimeric anti-Her2neuantibody ch4D5 and chimeric anti-FcγRIIB antibody ch8B5.3.4 (orhumanized anti-FcγRIIB antibody (h8B5.3.4)) in xenograft tumor model innude mice with adoptively transferred human monocytes as ADCC effectors.MBW (mouse body weight). ch4D5 ch8B5.3.4 Human ch4D5 at N297A at N297Aat IgG1 Tumor 4 μg/gm 4 μg/gm 4 μg/gm 4 μg/gm 8 cell s.c Monocytes ofmbw of mbw of mbw of mbw mice/group day 0 i.p at day 1 day 1 i.p day 1i.p day 1 i.p day 1 i.p A + — — — — — B + + — — — — C + + + — — —D + + + — + — E + + — — + — F + + — + — +

As shown in Table 10, 6 groups of 8 mice each are required for testingthe role of an anti-FcγRIIB antibody in tumor clearance with one targetand effector combination, with two different combinations of theantibody concentrations. These groups are A) tumor cells, B) tumor cellsand monocytes, C) tumor cells, monocytes, anti-tumor antibody, ch4D5, D)tumor cells, monocytes, anti-tumor antibody ch4D5, and an anti-FcγRIIBantibody, e.g., ch8B5.3.4, E) tumor cells, monocytes, and ananti-FcγRIIB antibody, e.g., ch8B5.3.4, and F) tumor cells, monocytes,ch4D5 N297A, and human IgG1. Various combination of antibodyconcentration can be tested in similar schemes.

Studies using the breast cancer cell line, SKBR-3, are carried out inparallel with the IGROV-1 model as SKBR-3 cells over-express Her2/neu.This will increase the stringency of the evaluation of the role ofanti-FcγRIIB antibody in tumor clearance. Based on the outcome of thetumor clearance studies with the IGROV-1 cells, modifications are madeto experimental design of future experiments with other targets.

The endpoint of the xenograft tumor model is determined based on thesize of the tumors (weight of mice), survival time, and histology reportfor each group in Table 10. Mice are monitored three times a week;criteria for growing tumors are abdominal distention and presence ofpalpable masses in the peritoneal cavity. Estimates of tumor weightversus days after inoculation is calculated. Based on these threecriteria from group D mice in Table 10 versus the other groups of micewill define the role of anti-FcγRIIB antibodies in enhancement of tumorclearance. Mice that show signs of visible pain or reach 5 grams oftumor weight are euthanized with carbon dioxide and autopsied. Theantibody-treated animals are followed for two months after thistime-point.

6.2.3.2 In Vivo Activity of FCγRIIB Antibodies in Xenograft Murine Modelwith Human Primary Ovarian and Breast Carcinoma Derived Cells

Primary tumors are established from primary ovarian and breast cancersby transferring tumors cells isolated from exudates from patients withcarcinomatosis. In order to translate these studies into the clinic, thexenograft model are evaluated with ascites- and pleural effusion-derivedtumor cells from two ovarian and two breast carcinoma patients,respectively. Pleural effusion, as a source of breast cancer cells, andimplantation of malignant breast tissue have been used to establishxenograft murine models successfully, see, e.g., Sakakibara et al.,1996, Cancer J. Sci. Am. 2: 291, which is incorporated herein byreference in its entirety. These studies will determine the broad rangeapplication of the anti-FcγRIIB antibody in tumor clearance of primarycells. Tumor clearance is tested using anti-tumor antibody, ch4D5 andanti-FcγRIIB antibody, e.g., ch8B5.3.4, in Balb/c nude mouse model withadoptively transferred human monocytes

Human ascites and pleural effusion-derived primary tumor cells. Ascitesfrom patients with ovarian cancer and pleural effusions from breastcancer patients are used. The ascites and pleural effusion from patientsmay contain 40-50% tumor cells and samples with a high expression ofHer2neu+ tumor cells will be used to establish the xenograft models.

Ascites and pleural effusion samples are tested for expression ofHer2/neu on neoplastic cells prior to establishment of the xenografttumor model. The percentage of the neoplastic cells versus othercellular subsets that may influence the establishment of the tumor modelwill be determined. Ascites and pleural effusion from patients withovarian and breast cancer, respectively are routinely analyzed todetermine the level of expression of Her2/neu+ on the neoplastic cells.FACS analysis is used to determine the percentage of Her2/neu+neoplastic cells in the clinical samples. Samples with high percentageof Her2/neu+ neoplastic cells are selected for initiation of tumors inBalb/c mice.

Histochemistry and Immunochemistry. Histochemistry andimmunohistochemistry is performed on ascites and pleural effusion ofpatients with ovarian carcinoma to analyze structural characteristics ofthe neoplasia. The markers that are monitored are cytokeratin (toidentify ovarian neoplastic and mesothelial cells from inflammatory andmesenchymal cells); calretinin (to separate mesothelial from Her2/neupositive neoplastic cells); and CD45 (to separate inflammatory cellsfrom the rest of the cell population in the samples). Additional markersthat will be followed will include CD3 (T cells), CD20 (B cells), CD56(NK cells), and CD14 (monocytes).

For immunohistochemistry staining, frozen sections and paraffinizedtissues are prepared by standard techniques. The frozen as well as thede-paraffinized sections are stained in a similar staining protocol. Theendogenous peroxidase of the tissues is quenched by immersing the slidesin 3% hydrogen peroxide and washed with PBS for 5 minutes. Sections areblocked and the primary antibody ch4D5 is added in blocking serum for 30minutes followed by washing the samples with PBS three times. Thesecondary anti-human antibody conjugated with biotin is added for 30minutes and the slides are washed in PBS for 5 minutes. Avidin-Biotinperoxidase complex (Vector Labs) is added for 30 minutes followed bywashing. The color is developed by incubating the slides in freshsubstrate DAB solution and the reaction is stopped by washing in tapwater. For H& E staining, the slides are deparaffinized and thenhydrated in different alcohol concentrations. The slides are washed intap water and placed in hematoxylin for 5 minutes. Excess stain isremoved with acid-alcohol, followed by ammonia, and water. The slidesare placed in Eosin and followed by 90 to 100% alcohol washes fordehydration. Finally, the slides are placed in xylene and mounted withfixative for long-term storage. In all cases, the percentage of tumorcells is determined by Papanicolaou stain.

Histochemical Staining. Ascites from two different patients with ovariancarcinoma are stained by Hematoxylin and Eosin (H & E) and Giemsa toanalyze the presence of tumor cells and other cellular types.

Murine Models. Samples from ovarian carcinoma patients are processed byspinning down the ascites at 6370 g for 20 minutes at 4 C, lysing thered blood cells followed by washing the cells with PBS. Based on thepercentage of Her2/neu+tumor cells in each sample, two samples, a medianand high expressor are selected for s.c inoculation to establish thexenograft model to evaluate the role of anti-FcγRIIB antibody, inclearance of tumors. It has been reported that tumor cells make up40-50% of the cellular subset of unprocessed ascites, and afterpurification ˜10-50×10⁶ tumor cells were obtained from 2 liters ofascites (Barker et al., 2001, Gynecol. Oncol. 82: 57-63). The isolatedascites cells are injected i.p into mice to expand the cells.Approximately 10 mice will be injected i. p and each mouse ascitesfurther passaged into two mice each to obtain ascites from a total of 20mice, which is used to inject a group of 80 mice. Pleural effusion ishandled in a manner similar to ascites and Her2neu+tumor cells areinjected into the upper right and left mammary pads in matrigel. Afters.c inoculation of tumor cells, mice are followed for clinical andanatomical changes. As needed, mice may be necropsied to correlate totaltumor burden with specific organ localization.

6.3 Screening of CD32B-Specific Monoclonal Antibodies

CD32B-specific antibodies will be screened for reactivity, raftassociation, CDC, and induction of apoptosis in B-cell lymphoma linesand cells from patients with B-cell malignancies. Isolation of cellsfrom patients and reactivity screening is described above.

Raft association. A measure of the ability of the antibody to triggerredistribution of the antigen into specialized membrane microdomains,lipid raft association is conveniently performed by measuring the amountof antibody recovered into the detergent-insoluble cellular fractionafter lysis with 0.5% TX-100 at 4C (Veri et al., 2001, Mol Cell Bio21:6939-6950; Cragg et al., 2004, Blood 103:2738-43). In a typicalexperiment, cells will be coated on ice with the antibody of interestand washed. An aliquot will be subjected to additional cross-linkingwith an appropriate secondary antibody. Pelleted cells will be subjectedto TX-100 detergent fractionation. Parallel samples will be solubilizedwith glucopyranoside, a detergent known to destroy lipid rafts, ordirectly with SDS-based Laemmli sample buffer to obtain the total amountof cell-associated antibody. The insoluble fractions will be analyzed bySDS-PAGE and western blot. Redistribution to lipid rafts with or withoutadditional cross-linking will be recorded by densitometric comparisons.

CDC. CDC will be assessed by one of several methods known in the art,such as propidium iodide (PI) exclusion in FACS analysis (Cragg et al.,2004, Blood 103:2738-43) or traditional radiolabel release (e.g., ⁵¹Crand ¹¹¹In release). In brief, cells will be incubated with titratingamounts of the antibodies of interest for 15 min at 37C followed by theaddition of serum (20% final concentration) as a source of complementand the incubation continued for additional 5 min prior to analysis.Owing to the high variation of human serum, Pel-Freeze rabbit serum willbe used as a standard source of complement. Pooled normal human AB serumwill also be prepared. Each batch of serum will be tested in red bloodcell lysis against rabbit serum for quality assurance.

Apoptosis. Apoptosis induced by soluble or plate immobilized anti-CD32Bantibodies will be studied by standard FACS-based methodology by usingannexin V membrane translocation and PI staining (Cragg et al., 2004,Blood 103:2738-43) in multi-color analysis to identify the population ofinterest (e.g. Cy5-CD19). Briefly, cells will be treated for differentintervals of time (2 to 18 hours) with titrating amounts of the antibodyof interest in free solution or immobilized on 96-well plates. Cellswill then be recovered by gentle scraping and/or centrifugation andstained with 1 ug/ml of FITC-annexin V plus 10 ug/ml of PI todistinguish between early apoptosis and secondary necrosis.

6.4 In Vivo Tumor Clearance Studies in Murine Tumor Xenograft Models ofLymphomas

The ability to prevent tumors in a mouse model of lymphoma is animportant criterion to determine the potential for an antibody toproceed into clinical studies.

A number of well characterized Burkitt's lymphoma cell lines areavailable for use as models of NHL (Epstein et al., 1966, J Natl CancerInst 37:547-559; Klein et al., 1968, Cancer Res 28:1300-1310; Klein etal., 1975, Intervirology 5:319-334; Nilsson et al., 1977, Intl J Cancer19:337-344; Ohsugi et al., 1980, J Natl Cancer Inst 65:715-718). Axenograft model of lymphoma formation has been established in nude micesimilar to previously reported models (Vallera et al., 2003, CancerBiother Radiopharm 18:133-145; Vuist et al., 1989, Cancer Res49:3783-3788).

In brief, the Burkitt's lymophoma cell line, Daudi (5-10×10⁶ cells),will be transplanted subcutaneously into an immunodeficient nu/nu mousestrain. The BALB/c nu/nu mouse strain will be used together withadoptively transferred human PBMC purified from a healthy donor aseffector cells. A prevailing effector cell population in human PBMC isrepresented by NK cells, which exert ADCC via their CD16A (FcγRIIIa). Anu/nu mouse strain in which the murine CD16A gene has been knocked outand which has been genetically engineered to express human CD16A willalso be used. This CD16A−/−huCD16Atg, nu/nu mouse allows for theexamination of anti-tumor activity in the context of a human Fc receptorwithout the need for the adoptive transfer of human cells.

Mice will be treated with the selected chimerized antibody injected i.p.on day 1, 4, 7, and 15. A starting dose of 4 ug/g of body weight will beused, but additional doses will be tested to establish the relativepotency of the antibodies in this model. Rituxan and Campath will beused for comparison. Furthermore, potential synergism of combinationtherapy with Rituxan or Campath will also be studied. In these studies,tumor growth and morbidity will be monitored to compare antibody treatedand control groups. Mice will be sacrificed immediately if moribund orat the completion of the studies. The tumors will then be excised andgross and microscopic necropsy performed. Cytopathology onparaffin-embedded sections and immunohistochemistry on frozen sectionswill be performed for a morphological and immunological evaluation ofthe tumor and cellular infiltrates.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

Various references are cited herein, the disclosure of which areincorporated by reference in their entirety.

1-22. (canceled)
 23. An isolated antibody, or an antigen-bindingfragment thereof, that binds human FcγRIIB, said antibody or fragmentthereof comprising three light chain complementarity determining regions(CDR) and three heavy chain CDRs from 8B5.3.4 antibody produced byhybridoma cell line having ATCC accession number PTA-7610, or aderivative thereof having no more than one amino acid substitution ineach of the three light chain CDRs and the three heavy chain CDRs. 24.The antibody or fragment thereof of claim 23, which specifically bindsto an extracellular domain of native human FcγRIIB with greater affinitythan with native human FcγRIIA.
 25. The antibody or fragment thereof ofclaim 23, wherein said antibody is the 8B5.3.4 antibody produced byhybridoma cell line having ATCC accession number PTA-7610.
 26. Theantibody or fragment thereof of claim 23, wherein said antibody is amonoclonal antibody, a single chain antibody, a bispecific antibody oris a humanized form of said 8B5.3.4 antibody.
 27. The antibody orfragment thereof of claim 23, comprising a heavy chain variable domainhaving the amino acid sequence of SEQ ID NO: 4 and/or a light chainvariable domain having the amino acid sequence of SEQ ID NO:
 3. 28. Theantibody or fragment thereof of claim 23, comprising an Fc domain thatcomprises at least one substitution in said Fc domain.
 29. The antibodyor fragment thereof of claim 28, wherein said at least one substitutionis at position 240, 243, 247, 255, 270, 292, 300, 305, 316, 370, 392,396, 416, 419, and/or 421 according to Kabat numbering.
 30. The antibodyor fragment thereof of claim 28, wherein said Fc domain has a leucine atposition 243, a proline at position 292, a leucine at position 300, anisoleucine at position 305, and a leucine at position 396 according toKabat numbering.
 31. The antibody or fragment thereof of claim 23,wherein said fragment is a F(ab′)2 fragment or a F(ab) fragment.
 32. Abispecific antibody comprising a first heavy chain-light chain pair thatcomprises the antibody or fragment thereof of claim 23, and a secondheavy chain-light chain pair that specifically binds a tumor antigen.33. A pharmaceutical composition comprising (i) a therapeuticallyeffective amount of said isolated antibody or fragment thereof of claim23; and (ii) a pharmaceutically acceptable carrier.
 34. A method ofenhancing antibody-dependent cell mediated cytotoxicity (ADCC) activityof an anti-cancer antibody or antigen-binding fragment thereof,comprising administering to a patient in need thereof: i) a firsttherapeutically effective amount of said anti-cancer antibody orfragment thereof; and ii) a second therapeutically effective amount ofan anti-FcγRIIB antibody or antigen-binding fragment thereof, comprisingthree light chain complementarity determining regions (CDR) and threeheavy chain CDRs from 8B5.3.4 antibody produced by hybridoma cell linehaving ATCC accession number PTA-7610, or a derivative thereof having nomore than one amino acid substitution in each of the three light chainCDRs and the three heavy chain CDRs.
 35. The method of claim 34, whereinthe anti-FcγRIIB antibody or fragment thereof specifically binds to anextracellular domain of native human FcγRIIB with greater affinity thanwith native human FcγRIIA.
 36. The method of claim 34, wherein theanti-FcγRIIB antibody is the 8B5.3.4 antibody produced by hybridoma cellline having ATCC accession number PTA-7610.
 37. The method of claim 34,wherein the anti-FcγRIIB antibody is a monoclonal antibody, a singlechain antibody, a bispecific antibody or is a humanized form of said8B5.3.4 antibody.
 38. The method of claim 34, wherein the anti-FcγRIIBantibody or fragment thereof comprises a heavy chain variable domainhaving the amino acid sequence of SEQ ID NO: 4 and/or a light chainvariable domain having the amino acid sequence of SEQ ID NO:
 3. 39. Themethod of claim 34, wherein the anti-FcγRIIB antibody or fragmentthereof comprises an Fc domain that comprises at least one substitutionin said Fc domain.
 40. The method of claim 39, wherein said at least onesubstitution is at position 240, 243, 247, 255, 270, 292, 300, 305, 316,370, 392, 396, 416, 419, and/or 421 according to Kabat numbering. 41.The method of claim 39, wherein said Fc domain has a leucine at position243, a proline at position 292, a leucine at position 300, an isoleucineat position 305, and a leucine at position 396 according to Kabatnumbering.
 42. The method of claim 34, wherein the anti-FcγRIIB antibodyor fragment thereof is a F(ab′)2 fragment or a F(ab) fragment.
 43. Themethod of claim 34, wherein the anti-cancer antibody or fragment thereofand the anti-FcγRIIB antibody or fragment thereof are comprised in abispecific antibody.